DNA sequencing methods and detectors and systems for carrying out the same

ABSTRACT

In some embodiments, an analyte detection system is provided that includes a nanochannel, an electrode arrangement, and a plurality of nanoFET devices disposed in the nanochannel. A plurality of nucleic acid base detection components can be used that include a plurality of nanopores, a plurality of nanochannels, a plurality of hybridization probes, combinations thereof, and the like. According to other embodiments of the present teachings, different coded molecules are hybridized to a target DNA molecule and used to detect the presence of various sequences along the target molecule. A kit including mixtures of coded molecules is also provided. In some embodiments, devices including nanochannels, nanopores, and the like, are used for manipulating movement of DNA molecules, for example, in preparation for a DNA sequencing detection. Nanopore structures and methods of making the same are also provided as are methods of nucleic acid sequencing using the nanopore structures. Surface-modified nanopores are provided as are methods of making them. In some embodiments, surfaced-modified nanopores for slowing the translocation of single stranded DNA (ssDNA) through the nanopore are provided, as are nanopores configured to detect each of a plurality of different bases on an ssDNA strand.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/984,269 filed Jan. 4, 2011, which claims the prioritybenefit of earlier filed U.S. Provisional Patent Applications Nos.61/291,950, 61/291,953, 61/291,964, and 61/292,061, all filed Jan. 4,2010, and each of which is incorporated herein in its entirety byreference.

FIELD

The present teachings relate to the field of DNA sequencing anddetectors useful for DNA sequencing. The present teachings also relateto the field of manipulating movement of DNA and other charged polymers,and systems for carrying out such movement. In addition, the presentteachings relate to the field of DNA detection using nanopores.

BACKGROUND

DNA sequencing genetic analysis methods have been complex, expensive,and lengthy. Detectors for carrying out DNA sequencing have beenexpensive and required optical components. Methods of sequencing nucleicacids have required many copies of a target nucleic acid strand to besequenced. A need exists for a less expensive, less time-consuming DNAsequencing method and for a less-expensive detector that does notrequire labeling of the DNA. A need also exists for a less complicated,less expensive DNA manipulation method and system and for a method andsystem for manipulating DNA molecules to achieve DNA sequencing.Moreover, a need exists for a nucleic acid sequencing method and systemthat is faster and that does not require substantial amplification of atarget strand to be analyzed.

SUMMARY

According to various embodiments, an analyte detection system isprovided that comprises a nanochannel having a first end, a second endopposite the first end, a top, and a bottom opposite the top. A pair ofelectrophoretic electrodes can be provided to move charged analytesthrough the nanochannel. The electrophoretic electrodes can comprise afirst electrophoretic electrode at the first end and a secondelectrophoretic electrode at the second end. A pair of orthogonalelectrodes can also be provided, comprising a first orthogonal electrodeat the top and a second orthogonal electrode at the bottom oralternately a first orthogonal electrode on one side and a secondorthogonal electrode on the other side. A further pair of orthogonalelectrodes can be provided in the axis not utilized by the two previouspairs of electrodes. Yet another option is to have the second and thirdelectrode pairs in the same plane. In some embodiments, the detector cancomprise, disposed in the nanochannel, a plurality of nano-field effecttransistor devices (nanoFETs). In some embodiments, one or more of thenanoFETs can comprise a vertical FET, for example, an FET arrangedvertically and/or comprising a q-tip shape comprising a gold-aluminumalloy tip, on a germanium layer, on a silicon post. The plurality ofnanoFETs can comprise at least four different nanoFETs eachfunctionalized with a different receptor analyte than the others. Insome embodiments, a target DNA molecule can be bound to a bead and thebead can be disposed in the nanochannel to hold the target moleculeduring a sequencing method. In some embodiments, an exonuclease enzymecan be bound to a bead and the bead can be disposed in the nanochannel.

According to various embodiments, a DNA sequencing system is providedthat comprises a plurality of nucleic acid base detection components anda memristor network. The memristor network can be in electricalcommunication with the plurality of detectors, and can comprise a3-dimensional network in some embodiments. The plurality of nucleic acidbase detection components can comprise a plurality of nanopores, aplurality of nanochannels, a plurality of hybridization probes,combinations thereof, and the like. In some embodiments, the pluralitynucleic acid base detection components comprises at least fourdetectors, and the four detectors can comprise a first detectorconfigured to detect adenine, a second detector configured to detectcytosine, a third detector configured to detect guanine, and a fourthdetector configured to detect thymine. In some embodiments, anadditional detector, or one of the four detectors can be configured todetect uracil. In other embodiments, an additional detector ordetectors, or one of the four detectors can be configured to detectother nucleosides such as inosine, or pseudouridine. In someembodiments, the detectors may be configured to detect any natural orsynthetic nucleic acid analog. In some embodiments, the detectors can beconfigured to detect proteins, RNA, carbohydrates, other biomolecules,or other molecules used as markers or labels, where the protein,carbohydrate, other biomolecules, or other molecule used as a marker orlabel is hybridized to, bound to or associated with a portion of asingle stranded or double stranded nucleic acid molecule. In someembodiments, the memristor network can comprise a memristor/transistorhybrid network. In some embodiments, memristors and/or memristor hybridcircuits perform real-time data analysis for multiple sensors atnanopore or nanochannel detection sites in a DNA sequencing system.

According to various embodiments of the present teachings, hybridizableoligonucleotides, also referred to herein as coded molecules, can behybridized to a target DNA molecule and used to detect the presence ofvarious sequences along the target molecule. For example, a target ssDNAmolecule can be contacted with a mixture of different coded moleculesand the reaction product can be detected using a nanopore, ananochannel, a combination thereof, or the like. The hybridizable codedmolecules can be selected and/or configured to affect ion current travelthrough a detector, for example, through an electrode pair pathway in ananopore detector. Each coded molecule that hybridizes can cause aelectrical signal through an electrical pathway, which signal can bedetected and used to reveal information about the target. Informationgathered from the signals detected can be used to determine portions ofthe sequence of the target and the positions of those portions along thelength of the target. In some embodiments, the signals associated witheach coded molecule may be unique, allowing direct identification of thecoded molecule. In other embodiments, there may be a pattern in theelectrical signal generated by coded molecules. The pattern may resultfrom different signals from different detectors, different signal levelsfrom the same detector, from detection from one or more detectorsdetermining that a coded molecule is not proximate to the detector, orinteracting with the detector or any combination thereof. A kitcomprising mixtures of coded molecules is also provided according tovarious embodiments of the present teachings, as are methods ofgenotyping using the kit. The kit can comprise the coded moleculescontained together or separately. The kit can also contain one or morestandards, reagents, buffers, combinations thereof, and the like.

According to various embodiments, the present teachings provide methodsof DNA sequencing and genotyping using the DNA sequencing systemsdescribed herein.

According to yet another aspect of the present teachings, devices,systems, and methods of manipulating a DNA molecule or other chargedpolymers are provided. In some embodiments, DNA can be manipulated forpositioning with respect to detectors, to enable DNA sequencing of thepolymer. In some embodiments, devices for orienting DNA molecules areprovided that comprise a nanopore or nanochannel.

According to various embodiments, a DNA molecule movement device isprovided that comprises a nanochannel having a first end, a second endopposite the first end, a top, and a bottom opposite the top. A pair oftranslation electrodes is also provided, comprising a first translationelectrode at the first end of the nanochannel and a second translationelectrode at the second end. At least three pairs of orthogonalelectrodes are provided, each pair comprising a first orthogonalelectrode at the top and a second orthogonal electrode at the bottom. Acontrol unit can be provided for individually controlling the voltageapplied to at least one electrode of each electrode pair. In someembodiments, the nanochannel is filled with an electrophoretic mediumand the pair of translation electrodes can comprise a pair ofelectrophoretic electrodes.

According to various embodiments, the present teachings provide methodsof DNA sequencing and genotyping using the DNA sequencing systemsdescribed herein.

According to various embodiments, DNA molecule manipulation systems areprovided for DNA sequencing. The systems can be useful for controllingthe movement and velocity of a DNA molecule during a sequencing method.In some embodiments, a DNA manipulation system is provided thatcomprises a nanochannel having a first end, a second end opposite thefirst end, a top, and a bottom opposite the top. A pair ofelectrophoretic electrodes can be provided to move charged analytesthrough the nanochannel. The electrophoretic electrodes can comprise afirst electrophoretic electrode at the first end and a secondelectrophoretic electrode at the second end. A pair of orthogonalelectrodes can also be provided, comprising a first orthogonal electrodeat the top and a second orthogonal electrode at the bottom. In someembodiments, the system can comprise, disposed in the nanochannel, aplurality of nano-field effect transistor devices (nanoFETs). Theplurality of nanoFETs can comprise at least four different nanoFETs eachfunctionalized with a different receptor analyte than the others. Insome embodiments, a target DNA molecule can be bound to a bead and thebead can be disposed in the nanochannel to hold the target moleculeduring a sequencing method. In some embodiments, an exonuclease enzymecan be bound to a bead and the bead can be disposed in the nanochannel.

According to various embodiments of the present teachings, a DNAmolecule manipulation device is provided that uses tunneling current asa detectable signal for determining individual nucleic acid bases of aDNA molecule. The devices can comprise built in redundancy features sobases can be read multiple times. In some embodiments, multipleelectrode structures are provided and in some embodiments the DNA ismoved with respect to the same electrode several times. In someembodiments, DNA is moved using an electric field or other means, andthen held in place utilizing an orthogonal electric field.

According to various embodiments, the DNA strand is stretched using abond or other mechanism at one or both ends of the DNA molecule, andthen a scan head is moved with respect to the bound DNA. In someembodiments, DNA is bound to a surface of a rigid structure on the scaleof the DNA strand, for example, bound to a nanotube or nanobead, andthen moved together with the structure past a fixed scan head using ananopositioning stage. In some embodiments, a pair or carbon nanotubesare arranged and separated by about the length of a single base, and DNAis caused to move through both of the carbon nanotubes, and thenanotubes are utilized as electrodes. In some embodiments, two nanotubesare oriented at right angles such that a DNA strand is positioned by onenanotube or nanopost and the base specific tunneling current is read bythe other nanotube.

According to various embodiments, the present teachings provide methodsof DNA sequencing and genotyping using the DNA sequencing systemsdescribed herein.

According to yet other various embodiments of the present teachings,nanopores are provided that can be useful for nucleic acid sequencing,as is a method for forming a nanopore structures. The method cancomprise treating a nanopore that is formed through a substratecomprising at least one layer of silicon or silica material. Thenanopore can comprise an inner sidewall having exposed silanol groups.In an alternative embodiment, a nanochannel may be utilized. The exposedsilanol groups can be reacted with a amino-containing compound such asan amino-containing alkoxysilane to convert the silanol groups toamino-containing functional groups. Then, the amino groups can bereacted with the copolymerization product of an acrylic ester ofN-hydroxysuccinimide and an acrylamide. The N-hydroxysuccinimde ester ofthe acrylic acid reacts with the amino group. In some embodiments, theacrylic acid ester of N-hydroxysuccinimide can be replaced with anacrylic acid of pentafluorophenol. The reaction results in covalentlybonding of a copolymerized product on the inner sidewall throughamidization. The resulting surface treatment polymer can be useful foraffecting the translocation rate of a ssDNA molecule through thenanopore, for stretching out the ssDNA as it passes through thenanopore, for imparting a preferential orientation to the ssDNA, tophysically confine the ssDNA to a region within the nanopore, todecrease the separation between the sensing element and the ssDNA, todecrease the effective size of the nanopore, thus allowing for largermanufacturing tolerances, a less demanding manufacturing process.Individual bases of the stretched out ssDNA can thus be more readilydetected by detection moieties in the nanopore, compared to whendetection of the bases in a non-stretched orientation.

According to various embodiments, the term “nanopore” as used hereinapplies also to the concept of a nanochannel. The term “nanopore” doesnot include any limitations as for geometry, aspect ratio, size, shape,cross-sectional profile, etc, other that a salient dimensioncharacterizing the geometry of the “nanopore” itself is smaller than 0.1microns. The nanopore can be “through” or “blind”, composed of a singleor more materials, arranged e.g. in layers, or others, each layer madeof one or more materials. The layer, as thin as a single atom, can be ofnon-constant thickness and depart from a substantially planar geometry.The electrically conductive material (“electrode”) can be flush withrespect to the local nanopore sidewall geometry, can protrude toward thecentral axis of the pore, or can be undercut in the peripheraldirection. The electrode layer does not need cover the entire plane, andonly a portion of the layer can be exposed inside the nanopore, in someembodiments. Multiple separate electrodes can be located on the samelayer and individual portions exposed separately on the surface of thenanopore.

According to various embodiments, a method is provided for surfacemodification of a nanopore through a substrate that comprises at leastone layer of a conductor, which may be a carbon nanotube, graphenelayer, InSnO, noble metal or noble metal alloy, used as an electrodelayer. The electrode layer can, for example, be electrically connectedto a voltage source and an applied potential can be used that causes theelectrode to act as an anode. At least a portion of an inner sidewall ofthe nanopore can be defined by an exposed surface of the at least onelayer. In some embodiments, the layer can comprise gold. According tovarious embodiments, the exposed noble metal or alloy thereof can bereacted, at the exposed surface thereof, with a thiolated compound, forexample, α-mercapto-polyol, such that a sulfur linkage to the exposedmetal surface is formed. The thiolated compound can also comprise aterminal nucleic acid base affinitive moiety that that can enable anon-covalent, physical, temporary, and reversible affinity. Althoughbinding may be referred to herein in this regard, it is to be understoodthat the selective association is non-covalent, physical, temporary, andreversible. Herein, “binding” can refer to a “non-zero/positiveaffinity,” the phrase “temporary binding” or can refer to affinitydriven interaction or sensing, of which actual binding is only one ofthe many possible interactions or sensing opportunities. Upontemporarily selectively associating to a complementary nucleic acidbase, the association can affect a current or voltage passing throughthe electrode. The change in current or voltage can then be detected andanalyzed to determine what type of base temporarily bound to the bindingmoiety. Furthermore, a change in electrical signal, for example, a DCsignal, an AC signal, or both, can be sensed, which results from avariation of any of a variety of electrically transducatable properties,for example, resistance, capacitance, inductance, polarization moment,tunneling current, and the like.

In some embodiments, a nanopore formed in a substrate is providedwherein the substrate comprises a plurality of spaced apart layers, eachcomprising a noble metal or noble metal alloy. In some embodiments, atleast one of the plurality of layers can comprise an exposed surfacethat has bonded thereto a first nucleic acid base binding (affinitive)agent. At least one different layer of the plurality of layers cancomprise an exposed surface that has bonded thereto a second nucleicacid base binding (affinitive) agent that is different than the firstone. Each of the first and second nucleic acid base binding (affinitive)agents can comprise, for example, a thiolated glycol comprising at leastone deoxyribonucleotide phosphate. The nanopore structure can beconfigured such that when the first or second nucleic acid base binding(affinitive) agent temporarily associates to a complementary base of anssDNA molecule passing through the nanopore, a change in current,voltage, or both, through the respective electrode, can be detected andused to identify the base temporarily bound.

According to various embodiments, a method is provided that comprisesforming a nanopore through a substrate that comprises at least one layerof graphene. The nanopore can comprise an inner sidewall, at least aportion of which comprises an exposed graphene surface. The exposedgraphene surface can be modified by a reaction that covalently bindsthereto a nucleic acid base binding (affinitive) agent. The binding(affinitive) agent can comprise a carbonyl linkage moiety and adeoxyribonucleotide phosphate. In some embodiments, the phosphate cancomprise a diphosphate or a triphosphate. In an alternative embodiment,the binding agent can be attached directly to the graphene, without alinkage group. In a further embodiment, the binding agent can consist ofa nucleobase, without the sugar or phosphate groups that are part of acustomary dNTP.

According to yet other embodiments of the present teachings, a nanoporeformed through a substrate is provided. The nanopore can comprise aninner sidewall and can have a diameter. The inner sidewall can besurface-modified to have chemically bound to the surface thereof apolymer extending radially inwardly, for example, toward the radialcenter of the nanopore. The polymer can extend inwardly by a distancethat is at least 25% of the length of the diameter, for example, about35% or about 45% of the length of the diameter. The diameter can be 100nm or less, for example, 20 nm or less, or 10 nm or less. The polymercan comprise any of the nanopore surface-modifying polymers describedherein.

In yet other embodiments of the present teachings, a multilayer nanoporeis provided, that is formed through a substrate. The nanopore cancomprise an inner sidewall defined, at least in part, by a first layer.The first layer can comprise an exposed surface at the inner sidewall.In some embodiments, the exposed surface can define an electrode, one ormore counter-electrodes, and one or more dielectrics that separate theelectrode from the one or more counter-electrodes. In some embodiments,at least two counter-electrodes are defined at the nanopore innersidewall and each can be surface-modified with a different nucleic acidbase binding (affinitive) agent covalently bonded thereto at the exposedsurface. With such a configuration, each of the two different nucleicacid bases can be identified by the first layer electrodes.Configurations having multiple different layers of electrodes can beused to detect all possible nucleic acid bases and/or to providedetection redundancies useful to verify results. These and other aspectsof the present teachings will be more fully understood with reference tothe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a detection scheme according tovarious embodiments of the present teachings wherein DNA Sequencing iscarried out using four serial arranged nanoFET stacks, each stackcomprising four nanoFETs functionalized with G, A, T, and C receptormolecules, respectively.

FIG. 2 is a schematic illustration of a detection system according tovarious embodiments of the present teachings and comprising anelectrophoretic channel, a plurality of functionalized nanoFETs arrangedin the channel, electrical circuitry, and a DNA target molecule to besequenced in the nanochannel.

FIGS. 3A-3B schematically illustrate a method of preparing beads,enzymes, and target DNA to be used in a nanoFET chip for DNA sequencing,according to various embodiments of the present teachings.

FIG. 4 is a schematic illustration of a nanopore detection system, atarget molecule in the form of a single stranded DNA scaffold, and aplurality of different hybridizable coded molecules hybridized tovarious portions of the DNA scaffold, forming double-stranded segmentsalong the scaffold.

FIGS. 5A-5C depict a DNA molecule manipulation nanochannel showing threepairs of electrodes, wherein the electric field phase and the DNAspacing is in perfect alignment (FIG. 5A), out of alignment (FIG. 5B),and in tuned alignment (FIG. 5C), according to various embodiments ofthe present teachings.

FIGS. 6A-6C depict a DNA molecule manipulation nanopore showing threepairs of electrodes, wherein the electric field phase and the DNAspacing is aligned (FIG. 6A), out of phase (FIG. 6B), and re-aligned(FIG. 6C), according to various embodiments of the present teachings.

FIG. 7 depicts a DNA molecule manipulation nanopore showing three pairsof electrodes, a pair of translation electrodes, and a potentialbarrier, according to various embodiments of the present teachings.

FIGS. 8A-8C depict a DNA molecule manipulation nanopore two phase deviceshowing three pairs (one set) of electrodes in one layer and three pairs(one set) of electrodes in another layer, wherein the two sets are outof phase with each other (FIG. 8A), the two sets are out of phase witheach other (FIG. 8B), and the two sets are re-aligned by a change infield strength (FIG. 8C).

FIGS. 9A-9C are a top view, side view, and end view, respectively, of aDNA molecule manipulation channel showing electrode arrangements and anelectrode gap, according to various embodiments of the presentteachings.

FIGS. 10A-10C are a top view, side view, and end view, respectively, ofa DNA molecule manipulation channel showing electrode arrangements and atunneling electrode, according to various embodiments of the presentteachings.

FIGS. 11A-11C are a top view, side view, and end view, respectively, ofa DNA molecule manipulation channel showing electrode arrangements and acorner electrode, according to various embodiments of the presentteachings.

FIGS. 12A-12C are a top view, side view, and end view, respectively, ofa DNA molecule manipulation channel in the form of a serpentine channel,and showing electrode arrangements and tunneling electrodes, accordingto various embodiments of the present teachings.

FIGS. 13A-13C are a top view, side view, and end view, respectively, ofa DNA molecule manipulation binding trough and an atomic forcemicroscope positioned for scanning the trough, according to variousembodiments of the present teachings.

FIGS. 14A-14C are a top view, side view, and end view, respectively, ofa DNA molecule stretching structure and electrode arrangement, accordingto various embodiments of the present teachings.

FIG. 15 is a side view of a dual-nanotube configuration according tovarious embodiments of the present teachings, wherein a DNA molecule isstretched through two carbon nanotubes that function as tunnelingelectrodes, the distance of a single base of DNA separates thenanotubes, and a tunneling current between the tubes is used tocharacterize the isolated base in the gap.

FIG. 16 is a side view of a dual-nanotube configuration according tovarious embodiments of the present teachings, wherein a DNA molecule isstretched through a movable nanotube tip and another carbon nanotubecomprises a fixed post, a gap is configured between the fixed nanotubepost and the moveable nanotube tip, the nanotube tip approaches the postfrom the side, and a tunneling current between the tubes is used tocharacterize the isolated base in the gap.

FIG. 17 is a side view of a fixed post nanotube and atomic forcemicroscope (AFM) tip configuration according to various embodiments ofthe present teachings, wherein a DNA molecule is stretched around thefixed post nanotube and the gap between the fixed post nanotube and theAFM tip is configured to present just one base at-a-time to the AFM tip.

FIG. 18 is a schematic illustration of a detection scheme according tovarious embodiments of the present teachings wherein DNA Sequencing iscarried out using a corner structure and a nanobud, and in the exampleshown, the nanobud is functionalized with nucleic acid receptormolecule.

FIG. 19 is a schematic illustration of a cross-sectional side view of anssDNA molecule being moved through a nanopore according to variousembodiments of the present teachings.

FIG. 20 is a top view of the nanopore shown in FIG. 19, taken throughline 2-2 of FIG. 19, and depicting the physical pore diameter and theeffective pore diameter.

FIG. 21 is a schematic illustration of a cross-sectional side view of anssDNA molecule being moved through a nanopore according to variousembodiments of the present teachings, wherein the nanopore comprisesselective nucleic acid base binding (affinitive) agents bound tosurfaces of the electrodes.

FIG. 22 is a top view of the nanopore and molecule shown in FIG. 21,taken through line 4-4 of FIG. 21, showing an electrode andcounter-electrode configuration according to various embodiments of thepresent teachings.

FIG. 23 is a schematic illustration of a cross-sectional side view of anssDNA molecule being moved through a nanopore according to variousembodiments of the present teachings, wherein the nanopore comprisesselective nucleic acid base binding (affinitive) agents to bind with thefour different nucleic acid bases A, C, G, T, wherein the base binding(affinitive) agents are bound to four different respective electrodes.

FIG. 24 is a top view of the nanopore and molecule shown in FIG. 23,taken through line 6-6 of FIG. 23, showing two electrodes and onecounter-electrode in a configuration according to various embodiments ofthe present teachings.

FIG. 25 is a schematic illustration of a cross-sectional side view of anssDNA molecule being moved through a nanopore according to variousembodiments of the present teachings, wherein the nanopore comprisesindividual single layers of grapheme.

FIG. 26 is a schematic illustration of a graphene layer that can be usedas an electrode layer according to various embodiments of the presentteachings.

FIG. 27 is an enlarged view of a portion of FIG. 26, showing the averagedistance between nuclei in the graphene layer.

FIG. 28 is a cross-sectional side view of a nanopore surfacemodification method that can be used in preparing a nanopore accordingto various embodiments of the present teachings, wherein silanol groupson the exposed inner sidewall of a silica layer are subject to aminosilylation.

FIG. 29 is a cross-sectional side view of a nanopore surfacemodification method that can be used in preparing a nanopore accordingto various embodiments of the present teachings, wherein a trapping orentanglement copolymer is chemically bonded to the exposed inner surfaceof the nanopore shown in FIG. 28 by reacting the reaction product of anN-hydroxy succinimide ester of acrylic acid and N,N-dimethylacrylamidewith the amino groups on the exposed surface.

FIG. 30 is a cross-sectional side view of a nanopore surfacemodification method that can be used in preparing a nanopore accordingto various embodiments of the present teachings, wherein the trapping orentanglement copolymer on the inner sidewall of the nanopore shown inFIG. 29 is further capped with an amino-terminated polyol and/orcrosslinked with a diamino terminated polyol.

FIG. 31 is a cross-sectional side view of a nanopore surfacemodification method that can be used in preparing a nanopore accordingto various embodiments of the present teachings, wherein an exposedinner sidewall of a gold anode layer of a nanopore is subject to surfacemodification by reaction with a thiolated nucleic acid base binding(affinitive) agent.

FIGS. 32A-32F show the chemical structures of six respective thiolatedpolyols that can be used to conjugate a nucleic acid binding(affinitive) agent on the surface of a gold anode, according to variousembodiments of the present teachings.

FIGS. 33A-33D show the chemical structures of four respective nucleicacid binding (affinitive) agents that can be reacted with varioussugar/phosphate moieties and thiolated polyols as described herein toconjugate nucleic acid binding (affinitive) agents being bound by athiol linkage to the surface of a gold anode, according to variousembodiments of the present teachings.

FIG. 34 shows a deoxyribonucleotide triphosphate that can include one ofthe bases shown in FIGS. 33A-33D and may be used as a nucleic acidbinding (affinitive) agent according to various embodiments of thepresent teachings.

FIGS. 35 and 36 show a PNA moiety and a DNA moiety, respectively, thatcan be used in forming a nucleic acid binding (affinitive) agentaccording to various embodiments of the present teachings.

DETAILED DESCRIPTION

NanoFETs, Nanochannels, and Nanopores

According to various embodiments, an analyte detection system isprovided that comprises a nanochannel having a first end, a second endopposite the first end, a top, and a bottom opposite the top, oralternately a first orthogonal electrode on one side and a secondorthogonal electrode on the other side. A pair of electrophoreticelectrodes is provided, comprising a first electrophoretic electrode atthe first end and a second electrophoretic electrode at the second end.A pair of orthogonal electrodes is also provided, comprising a firstorthogonal electrode at the top and a second orthogonal electrode at thebottom. A further pair of orthogonal electrodes can be provided in theaxis not utilized by the two previous pairs of electrodes. Yet anotheroption is to have the second and third electrode pairs in the sameplane. Disposed in the nanochannel are a plurality of nano-field effecttransistor devices (nanoFETs) disposed in the channel. In someembodiments, one or more of the nanoFETs can comprise a vertical FET,for example, an FET arranged vertically and/or comprising a q-tip shapecomprising a gold-aluminum alloy tip, on a germanium layer, on a siliconpost. The plurality of nanoFETs can comprise at least four differentnanoFETs each functionalized with a different receptor analyte than theothers. In some embodiments, a target DNA molecule can be bound to abead and the bead can be disposed in the nanochannel to hold the targetmolecule during a sequencing method. In some embodiments, an exonucleaseenzyme can be bound to a bead and the bead can be disposed in thenanochannel.

According to various embodiments, a DNA sequencing device is providedthat comprises nanoFETs which have been functionalized to detect chargechanges on the surfaces of the nanoFETs. The surfaces of the respectivenanoFETs can be functionalized with analyte receptor moleculesexhibiting higher affinity to the intended analyte than the samenanoFETs would have without the analyte receptor molecules. In someembodiments, the receptor molecules can comprise nucleic acid basebinding moieties that can temporarily bind to bases of a target nucleicacid, for example, by hydrogen binding. Other binding moieties can beutilized. Such functionalized nanoFETs can be aligned in a sequentialmanner in a nanochannel as schematically shown, for example, in FIG. 1.In other embodiments, the detection can result from the interaction ofan electric field with molecule that has temporarily bound with thenanoFET. The field can be a DC field, an AC field or a combination of aDC and AC fields. In other embodiments, an electrode in close proximitycan functionalized with analyte receptor molecules exhibiting higheraffinity to the intended analyte than the same electrode would havewithout the analyte receptor molecules. In some embodiments, thereceptor molecules can comprise nucleic acid base binding moieties thatcan temporarily bind to bases of a target nucleic acid, for example, byhydrogen binding. Other binding moieties can be utilized. In yet otherembodiments, both the nanoFET and the electrode can be functionalized.In some embodiments, the same moiety can be used to functionalize thenanoFET and the electrode. In other embodiments, different moieties canbe used to functionalize the nanoFET and the electrode. In someembodiments, an electrode in proximity to a nanoFET can be within 1 nmof the nanoFET. In other embodiments, the electrode in proximity to ananoFET can be from 1 nm to 10 nm from the nanoFET. In yet otherembodiments, an electrode in proximity to a nanoFET can be from 10 nm to100nm from the nanoFET.

As illustrated in FIG. 1, analytes can be made to migrate through thenanochannel by applying an electric field and temporarily binding theanalytes to the functionalized surfaces of the modified nanoFETs. Thetemporary binding can cause a unique physical signal such as a specificconductance. Other methods of transport can be used and include, forexample, fluid flow, centripetal force, combinations thereof, and thelike. In some embodiments, a nanoFET A is functionalized with a receptormolecule for A, a nanoFET B is functionalized with a receptor moleculefor B, a nanoFET C is functionalized with a receptor molecule for C, anda nanoFET D is functionalized with a receptor molecule for D. A singledetection module for A, B, C, and D can be constructed by aligning theA, B, C, and D nanoFETs. In some embodiments, a plurality of detectionmodules can be serially connected to increase the detection accuracybecause the same analyte can be made to bind with multiple nanoFETsmultiple times. By using multiple nanoFETs of each type, the analytescan be detected multiple times, increasing sensitivity and accuracy. Insome embodiments, different numbers of nanoFETs may be used that arefunctionalized with different molecules. As a nonlimiting example, if Ais more difficult to detect than B, C or D, more nanoFET detectors maybe functionalized with A than the number of nanoFET detectorsfunctionalized with B, C or D. Any combination and any order offunctionalized detectors may be utilized. In some embodiments, anycombination and any order of differently functionalized electrodes thatare in proximity to a nanoFET may be utilized. In other embodiments, anycombination and order of similarly or differently functionalizedelectrodes and nanoFETs may be utilized, where each of the electrodesand the nanoFETs that are in respective proximity may be functionalized.In other embodiments, any combination and order of similarly ordifferently functionalized electrodes and nanoFETs may be utilized,where some of the electrodes and the nanoFETs that are in respectiveproximity may be functionalized.

The device and method can be used for any analyte detection, forexample, by attaching appropriate different receptor molecules. DNAsequencing is used herein to simply exemplify, not limit, the presentteachings. In some embodiments, the receptor molecules can comprisebases that are complementary to the analyte bases to be detected, forexample, T for A, A for T, C for G, and G for C. In some embodiments,the receptor molecules can comprise synthetic receptors such as PNAs orother non-DNA receptors. In some embodiments, an additional detector, orone of the four detectors can be configured to detect uracil. In otherembodiments, an additional detector or detectors, or one of the fourdetectors can be configured to detect other nucleosides such as inosine,or pseudouridine. In some embodiments, the detectors may be configuredto detect any natural or synthetic nucleic acid analog. In someembodiments, the detectors can be configured to detect proteins, RNA,carbohydrates, other biomolecules, or other molecules used as markers orlabels, where the protein, carbohydrate, other biomolecules, or othermolecule used as a marker or label is hybridized to, bound to orassociated with a portion of a single stranded or double strandednucleic acid molecule. In other embodiments, the analyte detectionsystem can be used to determine the sequence or partial sequence of aprotein, by the use of appropriate analyte receptor molecules.

In some embodiments, the nanoFETs can be fabricated using nanowiretransistors, carbon tube transistors, graphene transistors, or othermore standard semiconductor-based transistors. In some embodiments,additional amplification of the current from the nanoFET can beperformed adjacent the nanoFETs to minimize noise. In some embodiments,an amplifier such as a voltage amplifier, a current amplifier, anintegrator, a combination thereof, and the like, can be used. In someembodiments, an amplifier that performs additional amplification, whichis adjacent to the nanoFET can be made as part of the same set ofprocesses in which the nanochannel and or nanoFET is fabricated. In someembodiments, an amplifier that performs additional amplification, whichis adjacent to the nanoFET can be made as part of a different set ofprocesses from those used to fabricate the nanochannel and or nanoFET,but may be still part of the same structure. In other embodiments, thean amplifier that performs additional amplification, which is adjacentto the nanoFET can be made as part of a separate structure. The separatestructure can be a printed circuit board, a hybrid circuit.

According to various embodiments, a DNA molecule can be digestedsequentially by an exonuclease enzyme to form nucleotide monophosphateproducts that are negatively charged. In an exemplary embodiment, theproducts can be introduced into the nanochannel, or to a nanopore, whichis embedded with nanoFETs functionalized with receptor molecules for G,A, T, C, and/or other nucleosides. The binding time duration can betuned by tuning the electric-field strength generated by theelectrophoretic electrodes or by tuning the affinities of the receptormolecules immobilized on the nanoFET. Sequence information is the mostimportant information in DNA sequencing, so the binding can be atransitory event, which can be used to prevent phasing errors of thesequence information.

To achieve efficient detection, the nucleotides can be focused on thenanoFET by applying an electric-field in directions other than theflowing direction. FIG. 2 shows an exemplary arrangement whereinorthogonal electrodes are arranged to pull negatively charged productanalytes down to the bottom of a nanochannel 10, and electrophoreticelectrodes are arranged at opposite ends of nanochannel 10 to move theproducts through the nanochannel and across four different nanoFETs 12,13, 14, and 15. NanoFETS 12, 13, 14, and 15 can be functionalized asdescribed herein or with nucleic acid base binding agents comprisingnucleic acid bases and linkage moieties, for example, a thiolated diollinkage to a gold anode nanoFET surface. As shown in FIG. 2,counter-electrodes 18, 20, 22, and 24 can be provided to form electrodepairs with nanoFETs 12, 13, 14, and 15, respectively. In someembodiments, this is achieved by creating a very small channel throughwhich the dNTPs can move, for example, channels as small as 5 nm×100 nm.In other embodiments, the width of the nanochannel can be wider than 5nm, where one of the nanoFET or electrode is configured on one wall ofthe nanochannel, while the other of the nanoFET or electrode can beconfigured to be on the tip of an AFM (Atomic Force Microscope). The tipof the AFM can then be configured to be placed within the nanochannel.The tip of the AFM can be configured to be adjustable with respect tothe one of the nanoFET or electrode on the wall of the nanochannel.Multiple AFM tips can be used, where a number of the tips can beadjusted together, for example two, four, eight or more tips can beconfigured to be adjusted together. Alternatively, each of the multipleAFM tips can be individually adjustable. In other embodiments, thenanoFET can be replaced with another electrode, such that the twoelectrodes can be configured in a pair to measure a tunneling currentthrough the sample molecule. One or both of the tunneling currentelectrodes can be functionalized with moieties that can interact withthe sample molecule. The two electrodes in the pair of tunnelingelectrodes can be functionalized with the same moiety, or with differentmoieties on each of the electrodes in the tunneling electrode pair.

In some embodiments, an electric field can be applied orthogonal to theplane in which the nanoFETs are placed to insure that they will interactwith the receptor molecules on the nanoFETs. In some embodiments, thefield responsible for transporting the dNTPs through the nano-channelcan be removed so that there is sufficient time for interaction andmeasurement. In some embodiments, the orthogonal field can beoscillated, for example, to be synchronous to changes in theelectrophoretic field to permit interactions with the affinity moleculesattached to the nanoFETs, or tunneling electrodes. In some embodiments,the orthogonal field can be modulated to change the rate of binding orunbinding of the nucleotides. In some embodiments, the orthogonal fieldcan be configured to be at a frequency that is resonant with anoscillation of a portion of the sample molecule. In some embodiments,the frequency of the orthogonal field can be changed over a range, suchthat a difference in the detected tunneling current or current in thenanoFET can be determined between an intended portion of the samplemolecule or analyte which has bound to one of an electrode, tunnelingelectrode or nanoFET, due to the higher affinity of the receptorassociated with the electrode, tunneling electrode, or nanoFET and theintended analyte than for other unintended portions of the samplemolecule or analytes. In some embodiments, the difference in detectionmay result from a change in the amount of tunneling current or currentthrough the nanoFET. In other embodiments, the difference may result ina change in the phase of the tunneling current or current through thenanoFET. In yet other embodiments, the detected difference can resultfrom a combination of the amount and the phase of the tunneling currentor current through the nanoFET. Temperature, buffer composition, and thelike, can be controlled to provide appropriate binding times. In someembodiments, the temperature can be cycled to provide controlled bindingand unbinding. In some embodiments, a second orthogonal pair, or groupof electrodes may be used, where the second pair of orthogonalelectrodes may be used to position the sample molecules in the axisorthogonal to the electrophoretic electrodes. This second orthogonalpair of electrodes can be used to position the sample molecule. Thepositioning of the sample molecule or analyte(s) can result in improvedopportunity for binding, or interaction between the functionalizedelectrode, tunneling electrode, or nanoFET than would exist without thepresence of the field resulting from the second orthogonal pair or groupof electrodes. The improved binding or interaction between the samplemolecule or analyte(s) can result in improved detection from thetunneling current or current in the nanoFET.

FIGS. 3A-3B schematically illustrate a method of preparing beads to beused in a nanoFET chip for DNA sequencing, according to variousembodiments of the present teachings. The method can be used to preparebeads useful in a device such as the device shown in FIG. 2. As shown inFIGS. 3A-3B multiple exonuclease enzymes can be tethered to beads, atarget DNA can be hybridized to an attachment site on the beads, and thebeads can be used to hold the target in the device.

As an exemplary nanoFET chip device operates, the exonuclease cleavesdNTPs one at a time from the target DNA and the cleaved dNTPs are causedto move across the functionalized nanoFETs or tunneling electrodes wherethe dNTPs are detected. In some embodiments, target DNA is preventedfrom being swept through the nano-channel by binding the target DNA to asubstrate. This can be done near an electrode that causes the dNTPs tobe swept through the nano-channel, or somewhere between the electrodeand the nanoFETs. Depending on the charge of the exonuclease enzyme, itmay be necessary to similarly bind it to the substrate through a linkerof sufficient length that it can interact with the target DNA. Therecould be several enzymes in the vicinity of each target DNA in order tominimize time for the enzyme action.

In some embodiments, the voltage associated with sweeping out thereleased dNTPs into the nanochannel can be removed or modulated in orderto permit interaction between the target DNA and the tetheredexonuclease enzymes. In some embodiments, other means of changing thespeed of the sample molecules can be utilized. This part of the devicecan be in an area where there is no orthogonal field so as to preventinteraction between the target DNA and the exonuclease. In someembodiments, both the target DNA and the exonuclease enzyme can beremoved and replaced. Attachment can be effected by utilizing ligatedprimers of DNA PNA, or utilizing nonspecific primers. In someembodiments, other methods of attachment such as using Biotin andStreptavidin can be used. In some embodiments, the target DNA can haveone strand protected from activity by the exonuclease, such that thesecond strand can be synthesized by an added polymerase, permittingrepeated degradation by an exonuclease enzyme, and subsequent repeateddetection of the DNA sequence. The target DNA can have a universalprimer ligated onto one end, with subsequent addition of the complementwhich may be added with the polymerase. Alternatively, the primer may bea hairpin primer, obviating the need for a second primer.

According to various embodiments, the device can have an array ofchannels to increase throughput. Target DNA can be attached to thesubstrate in such a way that a single target is associated with eachchannel; enrichment schemes such as that described in WO 2006/135782 canbe used to ensure odds better than would otherwise result from a Poissondistribution, and such reference is incorporated herein in its entiretyby reference. The channels can be fabricated in several different ways.In some embodiments, the transistors are fabricated on a planar surfaceand then a channel structure is created, for example, out of adielectric material. Polymethylmethacrylate (PMMA) can be used. In someembodiments, the channel is created out of silicon, for example, byetching utilizing natural crystal lines to create a V groove, orutilizing more traditional vertical etching. The fabrication can alsocomprise metallization, forming implants on the sides of the channel,and the addition of carbon nano-tube or nano-wire detector components.The channels can be physically separated by walls or passively separatedby an empty zone akin to having lanes on a gel.

According to various embodiments, the DNA sequencing system can comprisea plurality of nucleic acid base detection components and a memristornetwork. The memristor network is in electrical communication with theplurality of detectors, and can comprise a 3-dimensional network in someembodiments. In some embodiments, the memristor network can comprise amemristor/transistor hybrid network. The plurality of nucleic acid basedetection components can comprise a plurality of nanopores, a pluralityof nanochannels, a plurality of hybridization probes, combinationsthereof, and the like. In some embodiments, the plurality nucleic acidbase detection components comprises at least four detectors, and thefour detectors can comprise a first detector configured to detectadenine, a second detector configured to detect cytosine, a thirddetector configured to detect guanine, and a fourth detector configuredto detect thymine. In some embodiments, an additional detector, or oneof the four detectors can be configured to detect uracil. In otherembodiments, an additional detector or detectors, or one of the fourdetectors can be configured to detect other nucleosides such as inosine,or pseudouridine. In some embodiments, the detectors may be configuredto detect any natural or synthetic nucleic acid analog. In someembodiments, the detectors can be configured to detect proteins, RNA,carbohydrates, other biomolecules, or other molecules used as markers orlabels, where the protein, carbohydrate, other biomolecules, or othermolecule used as a marker or label is hybridized to, bound to orassociated with a portion of a single stranded or double strandednucleic acid molecule.

According to various embodiments, the present teachings provide a methodfor DNA sequencing using a DNA sequencing system as described herein.

In some embodiments, memristors and/or memristor hybrid circuits performreal-time data analysis for multiple sensors at nanopore or nanochanneldetection sites in a DNA sequencing system. In some embodiments,memristors and methods of using the same, that can be used according tothe present teachings, include those described, for example, in Strukovet al., The missing memristor found, Nature, Vol 453, May 1, 2008, inWilliams, How We Found the Missing Memristor, IEEE Spectrum, Dec. 11,2008, in Johnson, 3-D memristor chip debuts, EE Times Nov. 26, 2008, andin Eid et al., Real-Time DNA Sequencing from Single PolymeraseMolecules, published online in Science DOI: 10.1126/science. 1162986,Nov. 20, 2008. Each of these publications is incorporated herein in itsentirety by reference.

According to various embodiments, memristors, memristor/transistorhybrids, or combinations thereof, are used to collect and analyze datain real time from sensors at each of a plurality of nanochannels) ornanopore structures. In some embodiments, single, or multiple sensors inan array, are used to perform DNA sequencing. For the purposes of thisdisclosure “nanochannels” and “nanopores” are used interchangeably.Circuits constructed from such devices mimic aspects of the brain.Neurons are implemented with transistors, axons are implemented withnanowires in the crossbar, and synapses are implemented with memristorsat the cross points. In some embodiments, such a circuit can beconfigured to perform real time data analysis for multiple sensors. Insome embodiments, memristor crossbar memory cells are stacked on top ofa CMOS logic chip. Imprint lithography can be used to add a memristorcrossbar on top of a CMOS logic circuit. In some embodiments, anintegrated hybrid circuit is used that comprises both transistors andmemristors. Configuration bits can be located above CMOS transistors ina memristor crossbar. 3-D memristor chips comprisingtransistor/memristor hybrids can be used which have logic and density toperform significant real time data analysis of signals from multiplesensors, for example, multiple sensors at multiple nanopores,nanochannels, or other detectors.

An exemplary application within the scope of the present teachings isthe analysis of to real-time DNA sequencing data detected at a nanopore,nanochannel, or other detection component, where the properties of thememristor or a 3-D memristor/transistor hybrid are configured to handlemuch more data, and more efficiently, than conventional devices.According to the present teachings, the data can be stored in memory ina non-volatile manner. In some embodiments, real-time analysis of datacan be processed. The ability of memristors or memristor/transistorhybrids to act effectively in a neural network manner enables suchcircuits to learn and intervene in the DNA sequencing process to modifythe outcome of the DNA sequencing process and make it more effective.

According to various embodiments, long-term the neural networkingcapabilities of memristors memristor/transistor hybrids, transistors, ortunneling electrodes enable the monitoring of fluorescent emission andnon-fluorescent real-time DNA sequencing data, and can also learn. Suchnetworks can provide feedback to the sequencing system, change DNAsequencing parameters, and render the system more efficient. Forexample, read lengths can be improved through improved memristor,memristor/transistor hybrids, transistors, or tunneling electrodesfeedback and subsequent adjustments in the local detector environment.

According to various embodiments, real time data analysis by memristorsor memristor/transistor hybrids used in a neural network fashionprovides real-time feedback on the operation of one or more ZMWs orother detectors or detector components, to improve performance or alterprocesses and outcomes. Such systems can improve read length by openingor closing devices, adding chemicals at appropriate times, or carryingout other such operations. The memristors or memristor/transistorhybrids can be used to provide feedback real-time on data received, dueto their ability to form neural networks. According to the presentteachings, a network of nanopores, nanochannels, or other nucleic acidbase detecting components can be integrated with memristors ormemristor/transistor hybrids to form DNA sequencing systems that reportin real-time and that can tune themselves the more they are used, tocontinuously improve base detection. The emergent behavior can resultfrom the network processing more and more base calls and the memristorremembering the range of electrical signals detected for each of thefour different bases A, C, G, and T.

In some embodiments, the system can use logic to determine whether adetected electrical signal that falls somewhere between the strength ofa signal expected for a first base and the strength, duration or phaseof a signal expected for a second base, so that a reliable base call canbe made based on such an intermediate signal. In systems withredundancies, if the intermediate strength signal is later determined tohave come from a different base than the base previously called, thesystem can remember how to call a subsequent base that causes a similarintermediate strength, duration or phase of a signal. Other advantagesachieved from using memristors and memristor/transistor hybrids includethose described in the literature incorporated herein by reference.

According to various embodiments, non-volatile storage of fluorescenceemission data, ion current, tunneling current, nanoFET current, andother data obtained by multiple sensors at nanopores, or data from otherdetection devices can be obtained using memristors ormemristor/transistor hybrids. This storage can be useful in a regulatedclinical environment where the non-volatility of data can be importantfor legal reasons. The permanence of the memory is better in memristordevices than in most other electronic device memories.

According to various embodiments of the present teachings, hybridizableoligonucleotides referred to herein as coded molecules can be hybridizedto a target DNA molecule and used to detect the presence of varioussequences along the target molecule. For example, a target ssDNAmolecule can be contacted with a mixture of different coded moleculesand a signal resulting from an interaction with the reaction product canbe detected using a nanopore, a nanochannel, a combination thereof, orthe like. The hybridizable coded molecules can be selected and/orconfigured to effect ion current travel through a detector, for example,through an electrode pair pathway in a nanopore detector. Each codedmolecule that hybridizes can cause a unique electrical signal that canbe electrically, differentiated from other signals, and used to revealinformation about the target. Alternatively, tunneling current, orcurrent through a nanoFET may be used to create a differentiable signal.

Information gathered from the unique signals detected can be used todetermine a portion of the sequence of the target and the position ofthat portion along the length of the target. The result can be a strandof DNA that is single-stranded except along respective lengths wherecoded molecules hybridize. Each coded molecule can hybridize to arespective stretch of the DNA strand at a respective location that iscomplementary to a portion of the target. By detecting differentportions of the target in this manner, sequencing and/or genotyping canbe performed on the target. Although the system for carrying outgenotyping using such coded molecules may not necessarily be used tosequence a target in its entirety, and that a system using the codedmolecules might be more accurately described as a genotyping system, itis to be understood that such a system is also referred to herein as aDNA sequencing system.

An exemplary DNA sequencing system according to these embodiments willbe more fully understood with reference to FIG. 4. FIG. 4 is a schematicillustration of a nanopore detection system, a target molecule in theform of a single stranded DNA scaffold, and a plurality of differenthybridizable coded molecules hybridized to various portions of the DNAscaffold, forming double-stranded segments along the scaffold. As shown,coded molecules are assembled on a DNA scaffold into a linear array ofoligonucleotides (oligos). The different coded molecules can be selectedor made to have differential effects on ion current, tunneling current,or current through a nanoFET during traversal through the nanopore.Alternatively, in some embodiments, the detectors can be configured todetect proteins, RNA, carbohydrates, other biomolecules, or othermolecules used as markers or labels, where the protein, carbohydrate,other biomolecules, or other molecule used as a marker or label ishybridized to, bound to or associated with a portion of a singlestranded or double stranded nucleic acid molecule, or protein.

In FIG. 4, Address 1 refers to a specific DNA sequence. The codedmolecule identified as Address 1 Oligo is an oligo having a sequencethat is complementary to Address 1 so that Address 1 Oligo hybridizes toAddress 1. Address 1 Oligo. A unique signal or code can be generated byconfiguring Address 1 Oligo to exhibit a unique ion current effectduring traversal through the nanopore. As shown, Address 1 Oligocomprises a negatively charged DNA backbone, but it is to be understoodthat Address 1 Oligo could instead comprise a PNA backbone, a positivelycharged PNA backbone, or the like. If a set of different address oligosare used, they can be distinguished from one another and from stretchesof non-hybridized target due to the differences in ion current detectedthrough the nanopore. A negatively charged DNA address oligo exhibits adifferent current level in the measured circuit current than a neutralPNA address oligo. Likewise, a positively charged PNA would exhibit yeta third current level. Single-stranded stretches of DNA would exhibityet another different effect on ion current and can be used to furtherde-code the target. Yet further current levels could be generated as aresult of differences in current levels as a result of the detection ofproteins, RNA, carbohydrates, other biomolecules, or other moleculesused as markers or labels, where the protein, carbohydrate, otherbiomolecules, or other molecule used as a marker or label is hybridizedto, bound to or associated with a portion of a single stranded or doublestranded nucleic acid molecule, or protein. The different current levelscan be analogous to different colors in a fluorescent assay. In someembodiments, current levels can result from ion currents, tunnelingcurrents, currents in nanoFETs, or a combination of any of ion currents,tunneling currents, or currents in nanoFETs.

In some embodiments, ion current levels corresponding to single-strandedstretches of DNA are used as punctuation for the code. In someembodiments, valid codes could be followed by a distinctive currentlevel for a single-stranded segment, for example, followed by currentlevel indicative of a double-stranded segment. In some embodiments,valid codes could be followed by a distinctive current level for asingle-stranded segment, and then followed by current level indicativeof a single double-stranded segment. Detected codes that deviate fromthe pattern can be discarded as aberrant. In some embodiments, a firstset of current levels can be used for odd addresses and a second set ofcurrent levels can be used for even addresses. Such punctuation can beused to reduce the total number of codes that can be generated for afixed length of scaffold, and can serve as a quality control functionfor data analysis. In other embodiments, different types of currentdetection such as ion current, tunneling current or nanoFET current orany combination thereof can be utilized in a similar alternatingfashion, and may thus be used to reduce the number of codes, and serveas a quality control function for data analysis

In some embodiments, bulkiness is used as a property to affect ioncurrent through a nanopore and coded molecules comprising differentbulkiness can be used according to various embodiments of the presentteachings. Moreover, any chemical moiety that affects, or set ofchemical moieties that affect, ion current can be used in the codedmolecules according to various embodiments of the present teachings.Such chemical moieties can be part of the address oligo backbone,attached to the backbone, part of the bases used to specify thesequence, or attached to the bases.

In some embodiments, the address size can be in the range of from two to50 nucleotides (nt's), for example, from 4 to 30 nts, from 5 to 30 nts,or from 10 to 20 nts. The length of the address oligo can affect thelength of time that is used to acquire an unambiguous assessment of thecurrent level. The length of time correlating to the length of thecoding molecule can also be utilized to provide unambiguousdetermination of the type of coding molecule, or can be utilized toincrease the number of different codes available, or a combination ofdisambiguation and additional codes may be utilized. The length of timefor an address oligo or other label to pass through the nanopore candepend, for example, on oligo length and voltage bias across thenanopore. A lower voltage bias can provide more time to get an accuratecurrent measurement but also lowers the data collection rate. A lowervoltage bias also means a lower baseline current which can affect thenumber of different current levels that can be distinguished. Shorteroligos and higher voltage biases are advantageous for manufacturing,high data collection rate, and larger code space, for example, moredistinguishable current levels. Longer address oligos and lower voltagebias improves the quality of the current level data. The optimalselection for oligo length and voltage bias can be determinedempirically and/or experimentally. In other embodiments, the speed atwhich the sample molecule passes through the nanopore or nanochannel canbe affected by other parts of the structure.

According to various embodiments, address oligos can be used that haveaddresses on the scaffold right next to one another so that the finishedcoded molecule would be entirely double-stranded. A double strandedhybridized target could be used in an appropriately sized nanopore totraverse the nanopore in strictly a single file manner.

According to various embodiments, the coded molecules can bemanufactured by encapsulating scaffold DNA molecules in vesicles orhollow beads with a semi-permeable shell. The shell can be configured totrap the scaffold DNA but permit passage of one or more address oligos.In exemplary embodiments, the scaffold molecules can be retained basedon their length or due to attachment of a bulky or otherwise confiningmoiety. The beads can be large enough to encompass one million or morecopies of the target scaffold molecule, from about 1 to about 1000,000copies, or from about 1 to 10,000 copies. One, tens, hundreds,thousands, or millions of beads can be used together, or more.

In an exemplary embodiment, three types of address oligos are provided:DNA−, PNA0, and PNA+. In a first step, a collection of beads is dividedinto three pools. The first pool of beads is incubated with the DNA+oligos, the second pool is incubated with the PNA0 oligos and the thirdpool is incubated with the PNA+ oligos. After hybridization to address 1oligo is achieved, the address oligos are cross-linked to the scaffold.Cross-linking is effected such that the oligos do not exchange duringsubsequent manufacturing steps or when the coded molecule is used in anassay. Next, all the beads can be mixed together and re-divided intothree pools. Each of the pools can be incubated with one of the address2 oligos followed by cross-linking. The process can be repeated untilall the addresses are occupied. At the end, each bead contains acollection of coded molecules that all have the same code. To use acoded molecule collection, a bead can be broken or lysed open, a few ofthe molecules can be tested to determine the code, and the remainder ofthe molecules can be attached to the analyte-specific probes to be usedin an assay. In an exemplary embodiment, for SNP detection using aligation assay, the coded molecules can be attached to anallele-specific oligo, specific for a particular allele, for example,specific for a particular SNP.

According to various embodiments, the coded molecules can be made topass through a nanopore or nanochannel in a strictly single file manner.The order of the coded oligos along the scaffold can be maintained andrespective changes in the current single can correspond to the differentoligos that pass through the nanopore. In some embodiments, modifyingmoieties can be attached to the target so that one particular end of theoligo can be moved first through the opening to the nanopore. Such amarker or locating moiety can be used to orient a molecule to besequenced or genotyped. In other embodiments, a “drag chute” can beutilized such that the sample molecule is more likely to enter thenanopore or nanochannel in one direction. If order information ismaintained, oligos that elicit three different current levels in ascaffold with seven addresses generate 3⁷ or 2187 different codes. ForSNP analysis, two codes can be used per SNP. One code for each allelespecific oligo. Thus, three current levels with seven addresses wouldenable analysis of about 1000 SNP's in a single multiplex reaction. Forcertain voltage biases, about 300-400 microseconds can be used to detecta 10 kb double-stranded DNA molecule passing through a nanopore.Accordingly, one coded molecule can be detected every 1-10 millisecond.At one millisecond, this corresponds to 1,000 molecules per second or60,000 molecules per minute. For 2,000 different codes, 60,000 readsmeans that each code can be read approximately 30 times. This dataredundancy is sufficient to be statistically confident whether aspecific code is present or absent in a sample. Thus, the codedmolecules can be read from a 1000-SNP reaction in 1-10 minutes. In someembodiments, a higher voltage bias can be utilized until a detectablechange in one of an ion current, tunneling current, or nanoFET currentis detected, whereupon, either the voltage bias can be changed to permitadditional time for detection, or other means for changing the speed ofthe sample molecule through the nanopore or nanochannel can beimplemented.

According to various embodiments, 1,000 genotypes in 1-10 minutes can bebased on using a single detection channel. The detection apparatus cancomprise a simple device comprising a chamber and two electrodesseparated by a nanopore and relatively simple electronics that enable avoltage bias and a current measurement. In some embodiments, 10 or more,or 100 or more parallel channels or pores can be used such thatreactions at the 1000-plex level can be analyzed on 100 parallelchannels to generate detection throughput of 100,000 genotypes per 1-10minutes. In some embodiments, the voltage bias or other means ofchanging the speed of the sample molecule through the nanopore ornanochannel can be implemented so that individual pores or channels canhave the speed of their respective sample molecules modified.

In some embodiments, 10 different current levels are distinguished.Furthermore, lambda DNA can be used as a scaffold. In an exemplaryembodiment, 20 nt addresses are used and for a 50 kb lambda DNA molecule2500 addresses are provided. With 10 current levels, 10²⁵⁰⁰ possiblecodes are available. Unlimited numbers of codes are configurable.

According to various embodiments, detection across a nanopore isprovided by electron tunneling, functional electrodes, atomic forcemicroscopy, electrostatic force microscopy, combinations thereof, andthe like, for example, as described herein.

According to various embodiments, a large number of coded beads can besynthesized with minimal reagents, leading to lower manufacturing coststhan with individually coded bead synthesis. The length of linear codedmolecules can be increased to generate unlimited code space. Thedetection of coded molecules is faster than the detection of codedbeads, and coded molecules enable analyte assays in a homogeneousformat, leading to improved kinetics. For example, according to variousembodiments, ligation reactions occur more quickly in solution than theydo on the surface of a bead.

According to various embodiments, the methods of the present teachingsprovide assays having higher sensitivity for small amounts of analytes.There is no need to maintain an optical pathway, thus increasingflexibility in instrument design and facilitating designs with multipledetection channels. Current levels can be more discrete thanfluorescence emissions, leading to improved statistical power indiscriminating between signals.

According to various embodiments, methods using a set of coded moleculesas described herein exhibit very large code space, fast read times ofdiscrete signals using simple instrumentation, and efficientmanufacturing processes in enabling very sensitive, homogeneous analyteassays. In some embodiments, the present teachings can be implemented inconnection with a digital assay format. According to variousembodiments, the coded molecules of the present teachings, the methodsof using them, and the kits comprising them, are useful in manyapplications, including, for example, detecting SNPs, quantifying mRNA,genotyping, RNA expression assays, protein expression assays, smallmolecule quantification assays, applications outside the field of lifesciences, combinations thereof, and the like.

While exemplified with reference to nanopores, it is to be understoodthat the present teachings also encompass methods that use the codedmolecules in nanochannel detectors and in other DNA sequence detectors.

A kit comprising mixtures of coded molecules is provided according tovarious embodiments of the present teachings, as are methods ofsequencing and/or genotyping using the kit. The kit can comprise thecoded molecules contained together or separately. The kit can alsocontain one or more standards, reagents, buffers, combinations thereof,and the like.

The foregoing embodiments and variations thereof within the scope of thepresent teachings can be implemented in or with other systems, methods,and components for DNA sequencing. Exemplary teachings with which and inwhich the present teachings can be implemented, and which can beimplemented with and in the present teachings, include the systems,methods, and components described, for example, in Li et al., DNAmolecules and configurations in a solid-state nanopore microscope,Nature Materials, Vol. 2, pages 611-615 (September 2003), in U.S. Pat.No. 6,464,842 to Golovchenko et al., U.S. Pat. No. 6,627,067 to Brantonet al., U.S. Pat. No. 6,783,643 to Golovchenko et al., and in U.S.Patent Application Publications Nos. U.S. 2002/0187503 A1, U.S.2004/0229386 A1 to Golovchenko et al., U.S. 2008/0187915 A1 to Polonskyet al., U.S. 2006/0084128 to Sun, U.S. 2007/0190543 A1 to Livak, U.S.2007/0238186 A1 to Sun et al., U.S. 2008/0050752 to Sun et al., U.S.2009/0181381 A1 to Oldham et al., and U.S. 2009/0226927 to Sun et al.,each of which is incorporated herein in its entirety by reference.

Orientation of DNA for DNA Sequencing

According to various embodiments, the DNA molecule movement devicecomprises a nanochannel having a first end, a second end opposite thefirst end, a first side or top, and an opposite side or bottom oppositethe first side or top. The device comprises a pair of translationelectrodes comprising a first translation electrode at the first end ofthe nanochannel and a second translation electrode at the second end. Atleast three pairs of orthogonal electrodes are arranged with each paircomprising a first orthogonal electrode at the first side or top, and asecond orthogonal electrode at the opposite side or bottom.

In some embodiments, the device can be a part of a system that furthercomprises a control unit for individually controlling the voltageapplied to at least one electrode of each electrode pair.

In some embodiments, the nanochannel is filled with an electrophoreticmedium and the pair of translation electrodes can comprise a pair ofelectrophoretic electrodes.

According to various embodiments, an electric field control system isprovided wherein peaks of an electric field in a nanochannel can be madeto line-up with the periodicity of the DNA. Such fine tuning can enabledesired slowing and/or stopping of the DNA. The field can be tuned toprovide a net force on the DNA to slow it and/or stop it. FIGS. 5A-5Cdepict electrode arrangements and electric fields that can used tocontrol the movement of the DNA. In FIGS. 5A-5C, electrode pairs 91A and91B, 93A and 93B, and 95A and 95B are arranged along a DNA moleculemanipulation channel 97 and provide electric fields collectivelyillustrated as 99, having a phase. A DNA molecule 101 is moved throughthe channel. The fields shown are not accurate but are intended tocommunicate the general concept of field tuning according to variousembodiments.

In FIG. 5A, the phase of the electric fields and the DNA spacing arealigned and better controlled movement of the DNA target is enabled.According to various embodiments, an electric field control system canfinely tune the field to adjust for variations in dimensions,environmental conditions, spacing of the DNA, and the like variables.DNA spacing can change with electrophoretic field, pH conditions, andthe like, resulting in variability of phase with DNA spacing, asdepicted in FIG. 5B. In some embodiments, phase alignment can berectified by adjusting the voltage on one or more of the electrodepairs. In some embodiments, phase alignment can be rectified by directlymodulating the voltage of single electrodes. After such an in-place,pre-operational adjustment or fine tuning, the phase and DNA spacing canbe aligned, as shown in FIG. 5C.

In some embodiments, the migration rate of the DNA can be used as ameasurement method to tune the electrode fields to the spacing needed.In some embodiments, the DNA migration is minimized with optimal fieldlevels. The present teachings thus provide an on-the-fly tenability to aDNA molecule movement control system. The system can furthermorecomprise a reversible electrophoretic field such that a DNA molecule canbe made to traverse a nanochannel in a first direction, and then reverseits travel and traverse the nanochannel in an opposite direction. Therepeatability enabled can provide redundancies in DNA analysis andensure the accuracy of readings. The controlled movement can be useful,for example, in aligning a DNA molecule with a nanopore or nanochannelfor further processing therein.

According to various embodiments, the present teachings provide a methodfor DNA manipulation using a DNA molecule manipulation system asdescribed herein. According to various embodiments, the methods of thepresent teachings provide assays having higher sensitivity for smallamounts of analytes. There is no need to maintain an optical pathway,thus increasing flexibility in instrument design and facilitatingdesigns with multiple detection channels. Current levels can be morediscrete than fluorescence emissions, leading to improved statisticalpower in discriminating between signals.

While exemplified with reference to nanochannels, it is to be understoodthat the present teachings also encompass methods that use nanoporedetectors and other DNA base sequence detectors. According to variousembodiments, detection across a nanopore can be provided by electrontunneling, functional electrodes, atomic force microscopy, electrostaticforce microscopy, combinations thereof, and the like.

According to various embodiments of the present teachings, improvementsare provided to the devices, systems, and methods described in U.S.Published Patent Application No. U.S. 2008/0187915. For example, apotential barrier can be used in place of the well described, forexample, by applying a negative potential to the center electrodecompared to those at the sides. In such a device, the intensity of thetrapping energy is not affected, and therefore neither is the viabilityof the device. Instead, the result is an inconsequential shift of theequilibrium position of the charges by a half inter-polymeric unitdistance. In some embodiments, the barrier within a nanopore can becreated by any number of electrodes, including a single electrode.

According to various embodiments, and differently from the potentialwell, the flexibility of a polymer can be accounted for and theelectrical fields on the sides of the center electrode can be made tohave a positive effect. Due to the electrical forces directed away fromthe center electrode, DNA passing through the fields stretches and isheld under tension. Such tensional pre-loading is advantageous becausethe elongated DNA resembles more closely a rigid rod model and theinter-monomeric spacing useful for optimal operation of the device canbe more robustly reproduced. Furthermore, the tensional pre-loading alsoincreases the rigidity of the molecule and thus its stability, reducingdetrimental effects of the Brownian motion.

In some embodiments, the elasticity of a DNA molecule can be usedbeneficially to compensate for manufacturing tolerances which often donot provide exact spacing with precision. Under the ideal case of arigid rod, a dimensional mismatch would result in a reduction or zeroingof the trapping energy and, as a consequence, a reduction in theperformance of the device. In some embodiments, however, by virtue ofelasticity of a DNA molecule, and of the ability to control the pullingforce by adjusting the potential difference between pairs of electrodes,the two sides of an ssDNA molecule can be independently stretched tomatch the requirements imposed by the actual size of the differentlayers _(— —) According to various embodiments, a tuning process tomaximize a trapping energy, for example, to find an optimal potentialfor each electrode, is provided based on the actual geometry of thedevice. In some embodiments, a feedback process can be implementedwherein voltage imposed on one or more electrodes is varied, as shown inFIGS. 6A-6C based on the resulting change of a measurable electricalproperty sensed by the same electrode or sensed by one or more otherelectrodes or sensors.

In some embodiments, a potential barrier U is provided in the nanoporein place of a potential well. Such a barrier can be compatible with thesuperposition of an electrical field generated by the drag electrodes,as shown in FIG. 7. Although many combinations of potential profiles canbe used, with the potential distribution described herein and shown inFIG. 7, the entire length of a DNA molecule within the nanopore can beplaced under tension. It can be shown that the trapping action of thelocking electrical fields overcomes the translocational action of thedrag field. In a geometrically and electrically symmetrical case,specifically, wherein

-   -   V1=V3 and g1=g2        the result is that

${\frac{{V_{1} - V_{2}}}{{V_{t} - V_{c}}} > \frac{g}{p}},$where g is the gap between the electrodes and p the nominal pitchbetween unstretched monomeric units of the polymer.

In some embodiments, the analysis can easily be extended to thenon-symmetrical case, where p1 and p2 are the effective distances alongthe nanopore axis between monomeric units. In various embodiments, theeffective distances can be different as a result of the differentpre-tensioning of the two sides of the DNA molecule. The radialpositioning of the charged polymer, that is, the distance from the sidewalls, can be controlled by independently adjusting the biasing voltageof the potential distribution.

According to various embodiments, and similar to case with a potentialwell, the motion of a DNA molecule can be controlled by decreasing oreliminating the potential barrier for the amount of time necessary forthe DNA to move by a desired distance, before recreating the barrier. Anon-zero drag electrical field allows for imparting a preferentialdirection for the motion of the DNA once the locking action is removed.In the case of a single potential well, the lack of controllability inthe positioning of the DNA causes an inability to deterministicallyeffect its translocation, but such problems are overcome by the presentteachings.

In some embodiments, a potential barrier, a potential trap, or acombination of barriers and traps, can be cyclically shifted in time,spatially between different electrode pairs, with two electrodes being aminimum number of electrodes within the nanopore. As a result, a netmotion is provided to the DNA molecule as shown in FIGS. 6A-6C. Thepotential barrier can be gradually changed between different electrodepairs or sets, in much the same that the magnetic field is graduallyshifted in a stepper motor. Also, similar to a stepper motor, the pairsor sets of electrodes can be 180 degrees out of phase.

According to yet further embodiments of the present teachings, andsimilar to a stepper motor, additional pairs or sets of electrodes canbe used, for example five pairs or sets, or more, can be used, similarto a five-phase stepper motor. Although five are exemplified, any othernumber of pairs or sets of electrodes can be used. In such embodiments,the device can be compatible with both a symmetric geometry anddistribution of locking potentials, as well as with a geometrically andelectrically asymmetric configuration.

In some embodiments, electrodes which are in the same position axiallywith respect to a motion of a DNA molecule can have the same voltageimpressed upon them.

In other embodiments, electrodes which are in the same position axiallywith respect to the motion of the DNA molecule can have differentvoltages impressed upon them. As shown in FIGS. 8A-8C, the nanopore canhave multiple layers and there can be one, two, three, or moreelectrodes and/or electrode pairs, per layer.

While exemplified with respect to nanopores, it is to be understood thatsuch electrodes and arrangements can be configured as part of ananochannel.

The foregoing embodiments and variations thereof can be implemented inor with other systems, methods, and components for DNA manipulation,orientation, and/or sequencing. Exemplary teachings with which and inwhich the present teachings can be implemented, and which can beimplemented with and in the present teachings, include the devices,systems, methods, and components described, for example, in Li et al.,DNA molecules and configurations in a solid-state nanopore microscope,Nature Materials, Vol. 2, pages 611-615 (September 2003), in the articleof Ohshiro et al., Complementary base-pair-facilitated electrontunneling for electrically pinpointing complementary nucleobases, PNAS,Vol. 103, no. 1 (Jan. 3, 2006), in U.S. Pat. No. 6,464,842 toGolovchenko et al., U.S. Pat. No. 6,627,067 to Branton et al., U.S. Pat.No. 6,783,643 to Golovchenko et al., and in U.S. Patent ApplicationPublications Nos. U.S. 2002/0187503 A1, U.S. 2004/0229386 A1 toGolovchenko et al., U.S. 2008/0187915 A1 to Polonsky et al., U.S.2006/0084128 to Sun, U.S. 2007/0190543 A1 to Livak, U.S. 2007/0238186 A1to Sun et al., U.S. 2008/0050752 to Sun et al., U.S. 2009/0181381 A1 toOldham et al., and U.S. 2009/0226927 to Sun et al., each of which isincorporated herein in its entirety by reference.

Velocity Control of DNA Molecule Movement for DNA Sequencing

According to various embodiments of the present teachings, a DNAmolecule movement device is provided which may comprise a nanochannelhaving a first end, a second end opposite the first end, a top, and abottom opposite the top. The device may further comprise at least onepair of translation electrodes, comprising a first translation electrodeat the first end and a second translation electrode at the second end. Apair of orthogonal electrodes can also be provided, comprising a firstorthogonal electrode at the top and a second orthogonal electrode at thebottom. A control unit can individually control the voltage applied toeach of the electrodes. In some embodiments, each of the firsttranslation electrode and the second translation electrode eachcomprises an electrophoretic electrode, and the nanochannel can befilled, for example, with an electrophoretic medium. The control unitcan be configured to reverse a voltage across the pair of translationelectrodes, for example, to reverse a direction of movement of a DNAmolecule. In some embodiments, a pair of tunneling electrodes aredisposed in the nanochannel and configured to detect individual nucleicacid bases of a DNA molecule in the nanochannel.

According to various embodiments, a DNA molecule movement device isprovided that may comprise: a nanochannel having a first end, a secondend opposite the first end, a top, and a bottom opposite the top; a pairof translation electrodes, comprising a first translation electrode atthe first end and a second translation electrode at the second end; atleast one pair of orthogonal electrodes, each pair may comprise a firstorthogonal electrode at the top and a second orthogonal electrode at thebottom; and a control unit for individually controlling the voltageapplied to at least one electrode of each electrode pair. The controlunit can comprise a feedback sensor configured to sense feedback signalsrelated to DNA spacing and adjust the voltage, current, or both, appliedto at least one of the electrodes of the at least one pairs oforthogonal electrodes. Such control can be implemented on-site after aDNA sequencing system has been set-up for operation.

According to various embodiments, a method of controlling the movementof a DNA molecule through a nanochannel is provided. The methodcomprises providing a DNA molecule movement device which can comprise: ananochannel having a first end, a second end opposite the first end, atop, and a bottom opposite the top; a pair of translation electrodescomprising a first translation electrode at the first end and a secondtranslation electrode at the second end; at least one pair of orthogonalelectrodes, each pair may comprise a first orthogonal electrode at thetop and a second orthogonal electrode at the bottom; and a control unitfor individually controlling the voltage applied to at least oneelectrode of each electrode pair. The method can comprise: moving a DNAmolecule through the nanochannel; detecting DNA spacing during movementof the DNA molecule through the nanochannel, to produce a signal; andadjusting the voltage applied to one or more of the electrodes based onthe signal. The at least one pair of orthogonal electrodes can providean electric field phase, and adjusting the voltage can compriseadjusting the voltage to correlate the phase with the detected DNAspacing.

In yet other embodiments of the present teachings, a method ofcontrolling the movement of a DNA molecule through a nanochannel isprovided. The method uses a DNA molecule movement device comprising: ananochannel having a first end, a second end opposite the first end, atop, and a bottom opposite the top; a pair of translation electrodes,comprising a first translation electrode at the first end and a secondtranslation electrode at the second end; at least one pair of orthogonalelectrodes comprising a first orthogonal electrode at the top and asecond orthogonal electrode at the bottom; and a control unit forindividually controlling the voltage applied to at least one electrodeof each electrode pair. The method may comprise moving a DNA moleculethrough the nanochannel in a first direction, detecting nucleic acidbases of the DNA molecule during movement of the DNA molecule throughthe nanochannel, and reversing the voltage applied to the first andsecond translation electrodes to reverse the movement of the DNAmolecule to instead be in a second direction that is opposite the firstdirection.

According to various embodiments of the present teachings, a device isprovided as is shown in FIGS. 9A-9C. The device comprises a channel 130formed in any of a variety of ways. The fabrication approach can dependupon the method of fabrication of the electrodes to be used in thedevice. In the device shown in FIGS. 9A-9C, channel 130 can be etchedinto glass, milled into plastic, pressed into plastic, injection molded,micro-machined out of silicon, fabricated using semiconductor processessuch as photolithography or nanoimprinting, or made in a like manner.Translation electrodes 132 and 134 are used to cause a DNA molecule tomove from one end of channel 130 to the other end. Not shown is anyopening to permit introduction of the buffer, DNA, and/or cleaningsolutions into channel 130, or to remove DNA, buffer, or cleaningsolutions from channel 130. The channels 130 may be fabricated in aregular or irregular 1D or 2D array (not shown), and may have commonfeed lines (not shown) to permit introduction of the buffer, DNA, and/orcleaning solutions into channels 130, or to remove DNA, buffer, orcleaning solutions from channels 130. Common feed lines may be co-planarwith the array of channels, or may be fabricated in a parallel plane.Multiple layers of channels may also be fabricated, permitting a veryhigh density for detection. Voltage control of electrophoreticelectrodes may be common for all of an array, parts of an array, orindividual electrodes may be fabricated for each nanochannel. Additionalelectrodes may be utilized to concentrate sample at the inlet to ananochannel(s) relative to the common feed lines. Single electrode(s)per nanochannel(s) may be used in combination with one or more of theelectrophoretic electrodes. Alternatively, a pair (or more) ofelectrodes may be used for each nanochannel in order todielectrophoretically concentrate the sample at the inlet to thenanochannel(s). The dielectrophoretic electrodes may be usedindependently from the electrophoretic electrodes, or may be used inconjunction with one or more of the electrophoretic electrodes. It isalso possible to reverse the polarity of translation electrodes 132 and134, and or the electrophoretic electrodes, so that the DNA strand canbe re-read for better accuracy. Other approaches can be utilized inplace of translation electrodes to move the DNA, for example, lasertweezers (not shown). Movement can be manipulated in two axes. Arrays,either 2D or 3D, may utilized for any of the methods, systems or devicesdescribed herein, which control the movement, orientation, or detectionof molecules such as DNA.

Retention electrodes 136 and 138 are utilized to immobilize or slow theDNA during reading of the bases. Tunneling current electrodes 140 and142 are also provided. Retention electrodes 136 and 138 may cover, at aminimum, a large surface area in the area of tunneling electrodes 140and 142. When activated, they force the DNA towards the surface wheretunneling electrodes 140 and 142 are located, preventing or slowingfurther translation. They further reduce vibrational movement, and tendto orient the DNA, so that the base rotation between tunnelingelectrodes 140 and 142 is more consistent. Retention electrodes 136 and138 can be in direct contact with the buffer solution containing theDNA, and if this is the case, tunneling electrodes 140 and 142 can beseparated from retention electrodes 136 and 138 by a thin dielectric.Alternatively, it is possible to create an appropriate field byutilizing a thin dielectric between retention electrodes 136 and 138 andthe buffer, and if so, the field can be increased appropriately.

Tunneling electrodes 140 and 142 can be fabricated using micromachiningtechniques, but could also be etched after metal deposition and e-beamlithography. They are shown as being half the height of channel 130 butcan be shorter, for example, less than 100 nm, or taller, for example,the full depth of the nanochannel. Multiple sets of tunneling electrodescan be provided allowing reading at different points along the DNAstrand, at once, permitting faster reading of the strand and/or betterdata due to averaging data between different reads. As shown, aclearance 144 is provided above tunneling current electrodes 140 and142.

Another embodiment is shown in FIGS. 10A-10C, where reference numeralsthat are the same as used in FIGS. 9A-9C represent like elements. Theembodiment shown in FIGS. 10A-10C is different in how tunnelingelectrodes 146 and 148 are generated. Rather than having one electrodeon each side of the DNA, perpendicular to the backbone, tunnelingelectrodes 146 and 148 are created so that they cross the backbone onebase apart. This embodiment is simple to fabricate and more immune tonoise, as the two tunneling electrodes 146 and 148 are tightly coupled,rather than forming a comparatively large loop. Tunneling electrodes 146and 148 do not necessarily have to be a single base apart for theirentire length. Furthermore, an additional set of steering electrodes(not shown) can be provided on the walls perpendicular to tunnelingelectrodes 146 and 148, and can be used to center the DNA utilizingfeedback from tunneling electrodes 146 and 148. In some embodiments,upper retention electrode 136 can be replaced by a pair of steeringelectrodes, while retaining the functionality of both. In furtherembodiments, steering electrodes may be utilized on the same surfaces ofthe nanochannel as the retention electrodes, wherein the retentionelectrodes do not cover the entire surface. Multiple pairs of tunnelingelectrodes may be utilized at once, permitting faster reading of thestrand and/or better data due to averaging data between different reads.

In the embodiment shown in FIGS. 11A-11C, retaining electrodes may notbe used and instead a high frequency AC field is provided on thetranslation electrodes 132 and 134 to hold the DNA in one place. Thefield is also used to bring the DNA over into contact with cornertunneling electrode 150 as the DNA passes over a corner blockage 152.Corner tunneling electrode can be made to change frequency faster thanthe DNA can respond. This affect can be amplified by utilization of dragchutes or positively charged tails. In some embodiments, multiple cornerelectrodes can be created to permit multiple sets of tunnelingelectrodes and enable the benefits thereby accorded to detect the DNA atmultiple points at once, permitting faster reading of the strand and/orbetter data due to averaging data between different reads. Cornerelectrodes may be all on one side, or may alternate sides, causing theDNA to be stretched between the different corner electrodes. In analternative embodiment, retaining electrodes may be used in place of orin combination with the high frequency AC field to control the movementof the DNA.

FIGS. 12A-12C show an embodiment similar to that shown in FIGS. 11A-11CBut comprising a channel 160 in the form of a serpentine path instead ofa corner structure. Channel 160 may be of even width over its length andis shown as being sinusoidal, but can have other shapes, for example,with sharper turns. Tunneling electrodes 162 may be provided at the apexof the curves, or at other places along the curves. There may be onepair of electrodes for each curve, or there may be several pairs ofelectrodes for each curve.

FIGS. 13A-13C show an embodiment of a device 165 comprising a substrate166 having a trough 168 formed therein, wherein DNA is bound in thetrough. The DNA can be bound at both ends, or alternatively, at one endwhile the other end has a motive force applied to it such as an electricfield or laser tweezers. The DNA could be bound any of a variety ofmeans, including Biotin-Streptavidin and PNA-PNA hybridization.Stretching the DNA can be used to reduce vibrational noise. As shown theDNA is bound into the bottom of trench 168 at one end, and an electricfield is applied at the far opposite end and below the end of trench168, so that the DNA is forced into the bottom of trench 168. An AFM tip170 is configured for use as a tunneling electrode and is scanned alongtrench 168. Alternatively, the DNA can be translated by moving bothends, while the AFM tip moves only in one axis. This approach can useretaining electrodes so that the DNA is held and prevented fromrotating. In some embodiments, arrays of scanning AFM tips can be usedin a fashion similar to that currently used for large scale memorydevices.

FIGS. 14A-14C show an embodiment wherein a DNA molecule 172 is activelystretched between two structures that comprise DNA binding surfaces 174and 176. An electrode 178 is provided on both sides of the structure,one side of which is used as an AFM to determine the appropriate amountof force to apply. A second AFM is then utilized with tunnelingelectrodes 180 for determining the tunneling current. In someembodiments, the method can be performed in a vacuum, eliminatinginterference from buffer ions and water, and permitting the DNA to bedeeply cooled, further reducing vibrational noise. In some embodiments,arrays of scanning AFM tips can be used in a fashion similar to thatcurrently used for large scale memory devices.

According to various embodiments, another detection device that uses ascanning AFM approach determines the force of hybridization interactionfor a short PNA, for example, a 6 mer PNA, using a large number of tipsrepresenting an appropriate set of possible 6 mers. The AFM can beoscillated perpendicular to the DNA in order to maximize theinteraction. A map is then generated of hybridization force vs. positionvs. sequence to determine the statistically probable sequence. Differentlengths of binding moieties may be utilized, including 1, 2, 3, 4, 5, 7or more bases.

In some embodiments, different ones of the embodiments described hereinare combined, for example, by replacing the tunneling electrodesmentioned herein, with a scanning tip.

FIG. 15 is a side view of a dual-nanotube configuration according tovarious embodiments of the present teachings. A DNA molecule may bestretched through two carbon nanotubes 184 and 186 that function astunneling electrodes. The distance of a single base of DNA separatesnanotube 184 from nanotube 186, to define a gap 185. A tunneling currentbetween the tubes may be used to characterize a base isolated in gap185. Movement of DNA molecule 182 through nanotubes 184 and 186 can beeffected by translation electrodes 188 and 190. In this approach, DNAmolecule 182 is caused to flow through both nanotubes 184 and 186.Nanotubes 184 and 186 are then used as tunneling electrodes. Thediameter of each nanotube 184 and 186 may be optimized for singlestranded DNA. To optimize the reading, a rotating field can be appliedto the gap between nanotubes 184 and 186 to maintain rotationalconsistency of DNA molecule 182. The temperature of DNA molecule 182 canbe reduced, potentially significantly below OC in the nanotubes, whilestill maintaining nominally aqueous conditions. Motive forces for DNAmolecule 182 include electric fields but could also or instead includeprocessive enzymes such as polymerase or exonuclease. Additionalnanotubes may be utilized, wherein a DNA molecule may traverse seriallythrough additional nanotubes, permitting faster reading of the strandand/or better data due to averaging data between different reads. Thenanotubes may be configured to be located in a nanochannel as previouslydescribed; retaining and steering electrodes may be utilized to controlthe movement of the DNA into nanotubes(s).

FIG. 16 is a side view of a dual-nanotube configuration according tovarious embodiments of the present teachings, wherein a DNA molecule 192is stretched through a movable nanotube tip 194 and a second nanotubecomprises a fixed post 196. Each nanotube can independently comprise acarbon nanotube or a nanotube made from a different material. A gap 195is defined between fixed nanotube post 196 and moveable nanotube tip194. As movable nanotube tip 196 approaches the fixed nanotube post 194from the side, a tunneling current is provided between movable nanotubetip 194 and fixed nanotube post 196. The tunneling current can be usedto characterize a nucleic base of the DNA molecule, isolated in gap 195.Movement of DNA molecule 192 through nanotube 194 can be effected bytranslation electrodes 188 and 190.

As shown in FIG. 16, the gap can be configured between fixed nanotubepost 196 and moveable nanotube tip 194 that approaches fixed nanotubepost 196 from the side. DNA molecule 192 can be drawn through the gap byforce analogous to a rope around a pulley. A tunneling current betweenthe fixed nanotube post 196 and the movable nanotube tip 194 can be usedto characterize an isolated nucleic acid base located in the gap. DNAmolecule 192 can be drawn around the fixed nanotube post 196 by opticaltweezers, manipulating a bead, manipulating a magnetic bead, or thelike. For a 10 nm carbon nanotube fixed post the dimension is such thatonly one base is exposed to the movable nanotube tip 194, at a time. Insome embodiments, the fixed nanotube post diameter can be larger thanthe base spacing in single strand DNA. In some embodiments, the tip andpost are made of material other than carbon.

FIG. 17 is a side view of a device comprising a fixed post nanotube 100and an atomic force microscope (AFM) tip 102 configuration according tovarious embodiments of the present teachings. A DNA molecule 104 isstretched around fixed post nanotube 100 and a gap 106 is definedbetween fixed post nanotube 100 and AFM tip 102. Gap 106 is sized andconfigured to present just one base at-a-time to AFM tip 102. A bead 108can be attached to DNA molecule 104 and used to assist in manipulatingmovement of DNA molecule 104 through gap 106. Fixed nanotube post 100can be part of a conductor 110. In some embodiments, the fixed nanotubemay be affixed to a sharp corner or along a curve as shown in FIGS.11A-11C and 12A-12C. In some embodiments, nanobuds may be utilized tofurther localize the field and tunneling current. In other embodiments,one of the nanotubes may be configured to function as a nanoFET. Thenanotube may be further configured to have a nanobud in order to furtherlocalize the interaction between the DNA and the nanoFET.

FIG. 18 is a schematic illustration of a detection scheme according tovarious embodiments of the present teachings wherein DNA sequencing iscarried out using a corner structure and a nanobud. As shown in FIG. 18,the nanobud is functionalized with nucleic acid receptor molecule. As anssDNA molecule is manipulated around the corner structure, theindividual nucleic acid bases making up the ssDNA are exposed to thenanobud. In some embodiments, four corner structures with fourrespective, functionalized nanobuds can be provided to sense fourdifferent nucleic acid bases.

In some embodiments, other uses of the dual nanotubes can include, amongother things, observations of dye molecules, whether directly associatedwith DNA, or with other molecules. Nanotubes can be used to createnanoflow cells, and to cause lightwave concentration to be higher at thegap. Nanotubes with multiple carbon layers can be used in someembodiments. The gap between the dual nanotubes can function as a dipolenanoantenna to enhance single dye light emission according to a recentarticle by P. Muehlschlegel et al. Science 308, 1607 (2005), which isincorporated herein in its entirety by reference, and which describesusing dual metal nanorods as antennae.

In some embodiments, optimizing the use of a nanopore is provided bygenerating a rotational field on one side of a nanopore, which may stopor slow the progress of a DNA molecule through the nanopore. In someembodiments, the field strength can be reduced so that the DNA canproceed for a single base, and then the field strength is increasedagain. In some embodiments, an additional rotating field is created onthe opposite side of the nanopore, permitting higher field strength forbetter control of DNA molecule movement. In some embodiments, thisconcept is applied to the dual nanotube concepts described above.

According to various embodiments, an analyte detection system isprovided that comprises a nanochannel having a first end, a second endopposite the first end, a top, and a bottom opposite the top. A pair ofelectrophoretic electrodes is provided, comprising a firstelectrophoretic electrode at the first end and a second electrophoreticelectrode at the second end. A pair of orthogonal electrodes is alsoprovided, comprising a first orthogonal electrode at the top and asecond orthogonal electrode at the bottom. Disposed in the nanochannelare a plurality of nano-field effect transistor devices (nanoFETs)disposed in the channel. The plurality of nanoFETs can comprise at leastfour different nanoFETs each functionalized with a different receptoranalyte than the others. In some embodiments, a target DNA molecule canbe bound to a bead and the bead can be disposed in the nanochannel tohold the target molecule during a sequencing method. In someembodiments, an exonuclease enzyme can be bound to a bead and the beadcan be disposed in the nanochannel.

According to various embodiments, a DNA sequencing device is providedthat comprises nanoFETs which have been functionalized to detect chargechanges on the surfaces of the nanoFETs. The surfaces of the respectivenanoFETs can be functionalized with analyte receptor moleculesexhibiting higher affinity to the intended analyte than the samenanoFETs would have without the analyte receptor molecules. In someembodiments, the receptor molecules can comprise nucleic acid basebinding moieties that can temporarily bind to bases of a target nucleicacid, for example, by hydrogen binding. Such functionalized nanoFETs canbe aligned in a sequential manner in a nanochannel as schematicallyshown, for example, in FIG. 1.

Nanopore Structures and Methods of Nucleic Acid Sequencing Using theSame

According to various embodiments, the present teachings providefunctional nucleic acid base binding (affinitive) agents bound toelectrodes, to detect different nucleic acid bases along a target ssDNAstrand. Such detection does not need to use DNA base specific propertiessuch as tunneling current spectrum, and the like, for detectionspecificity. Instead a sensing element, for example, a polymer, ananowire, a nanotube, or the like may be used in some embodiments of thepresent teachings, which do not require detectors that rely on largesensitivities to changes of geometrical conformations to obtain ameasurable molecular and/or structural property. Instead, functionalchemical groups are attached to sensing elements, such as tunnelingelectrodes. The groups are specific to at least one of the differentbases of the ssDNA target. In some embodiments, the sensing element isdeformed by action of the moving ssDNA due to the affinity of itsfunctional group to at least a specific base. A base-specific measurablesignal can be produced from at least one electrode, or extracted fromthe analysis and or combination of the signals of 2 or more electrodes.In some embodiments, two or more sensing elements with differentbase-specificity can be integrated in the same layer of a nanoporestructure. Exemplary electrodes include those described, for example, inU.S. Pat. Nos. 7,619,290, 7,595,260, 7,500,213, 7,385,267, and7,301,199, which are incorporated herein in their entireties byreference.

In some embodiments, the nanopore can be formed in a substrate thatcomprises a plurality of spaced apart electrode layers each comprising anoble metal or an alloy thereof. In some embodiments, each electrode canindependently comprise a metal oxide, for example, indium-tin oxide(ITO), as materials for anodes. Other metal oxide surfaces, for example,comprising Al₂O₃, Ta₂O₅, Nb₂O₅, ZrO₂, TiO₂, or a combination or alloythereof, can also be used for chemically binding the affinitive agentsthrough phosphate or phosphonate groups. The different electrode layerscan be spaced apart from one another by intermediate insulatingdielectric, or semiconductor layers, or combinations thereof, includingcombinations of different materials within the same layer, for examplespaced apart by silicon nitride layers or silica layers. At least one ofthe electrode layers of the plurality can comprise an exposed surfacethat has bonded thereto a first nucleic acid base binding (affinitive)agent, and at least one different electrode layer of the plurality oflayers can comprise an exposed surface that has bonded thereto a secondnucleic acid base binding (affinitive) agent that is different than thefirst one. Each of the first and second nucleic acid base binding(affinitive) agents can comprise, for example, a thiolated polyolcomprising at least one deoxyribonucleotide phosphate. The nanoporestructure can be configured such that when the first or second nucleicacid base binding (affinitive) agent temporarily binds, i.e., ishybridized to, bound to, and/or associated with, a portion of the targetor sample molecule. The binding can be, for example, to a complementarybase of an ssDNA molecule passing through the nanopore. A change incurrent, voltage, or both, through the respective electrode, can bedetected and used to identify the base temporarily associated.

FIG. 19 is a schematic illustration of a cross-sectional side view of anssDNA molecule 21 being moved through a nanopore 23 according to variousembodiments of the present teachings. As shown in FIG. 19, nanopore 23comprises an inner sidewall 25 having bound thereto a trapping orentanglement polymer 26 adjacent an opening 28 of nanopore 23. Thetrapping polymer reduces the effective pore size of nanopore 23 and isuseful to comb, stretch, and/or tension ssDNA 21 as it moves throughnanopore 23. Trapping polymer 26 can be selectively immobilized to theinner sidewall of silica layer 32. Layer 32 can comprise silica orsilicon with silica as in silicon dioxide derived from a naturaloxidation of silicon on the exposed surface facing nanopore 23. Layer 32can be amorphous silicon deposited by CVD. The trapping polymer canconfine and tension ssDNA 21, can increase spatial sensing resolution atleast by reducing buckling of the ssDNA as it moves through nanopore 23,and can decrease the effective pore size of nanopore 23. Trappingpolymer 26 can comprise, for example, a dendritic polymer, a branchedpolymer, or a copolymer as described herein.

Nanopore 23 is formed in a substrate 30 comprising a first silica layer32, a first silicon nitride layer 34, a first electrode layer 36, asecond silicon nitride layer 38, a second electrode layer 40, a thirdsilicon nitride layer 42, a third electrode layer 44, and a secondsilica layer 46. Although three electrode layers 36, 40, and 44, aredepicted, more or less electrode layers can be used according to variousembodiments of the present teachings. In some embodiments, the structureis free of silicon nitride layer 34. In some embodiments, there is nosilicon nitride layer in between electrode layer 44 and silica layer 46.In some embodiments, the silicon nitride can be replaced by otherpolymer dielectrics, for example, polyimides or fluorinated poly(aryleneethers). Further details concerning the use of fluorinated poly(aryleneethers) can be more fully understood with reference to the article ofAldrich N. K. Lau et al., “Self-Crosslinkable Poly(arylene ether)sContaining Pendent Phenylenetriazene Groups,” J. Polym. Sci., Part A:Polym. Chem., 1994, 32, 1507-1521, which is incorporated herein in itsentirety by reference.

In some embodiments, nanopore 23 is formed in a substrate 30 comprisinga first electrically insulating layer 32 (e.g. silica), a secondinsulating layer 34 (e.g. silicon nitride), a first electrode layer 36,a third insulating layer 38 (e.g silicon nitride), a second electrodelayer 40, a fourth insulating layer 42 (e.g. silicon nitride), a thirdelectrode layer 44, and a last insulating layer 46 (for example, siliconnitride). Although three electrode layers 36, 40, and 44, are depicted,more or less electrode layers can be used according to variousembodiments of the present teachings.

As can be seen, each electrode layer has been surface-modified to have asensing polymer 48 (e.g. electrically conductive) attached thereto. Thesame or different polymers can be attached to the inner sidewallsurfaces of the three different electrode layers. In virtue of theconfinement induced by polymers 48, 48a, and 48 of the stretchinginduced by polymer 26, and on the spatial arrangement of the brushes ofpolymers 48, 48a, and 48b, the effective separation between theelectrode and the ssDNA molecule is reduced, thus originating a strongersignal and better resolution. It will be appreciated that the sensingpolymers 48, 48a, and 48b provide an effective solution to relay theelectrical signal between the respective electrodes and the ssDNAmolecule, the former being otherwise shielded by the electric diffuselayer existing on the nanopore surface. Furthermore, polymers 48, 48a,and 48b, actively contribute to control the positioning of the ssDNA byimpeding the lateral motion and dampen its Brownian motion, thusreducing the associated sensing noise. In addition, they allow for aconsistent orientation of the individual bases, with the added neteffect of further reducing the electrical noise associated with therandom distribution and motion of the bases.

As can be seen, each electrode layer has been surface-modified to haveattached thereto nucleic acid base binding (affinitive) agents 48. Thesame or different base binding (affinitive) agents can be attached tothe inner sidewall surfaces of the three different electrode layers. Asis shown at layers 36 and 44, certain bases of ssDNA molecule 21 aretemporarily bound (e.g., associated) to the nucleic acid base binding(affinitive) agents attached to electrode layers 36 and 44. Thetemporary binding (association) can be detected by a change in currentor voltage, for example, passing through the electrode. In someembodiments, different bases on the ssDNA react with different basebinding (affinitive) agents to produce different changes in currentwhich can be used to detect the type of base temporarily bound at therespective electrodes.

In some embodiments, the exposed portion of each layer can have adifferent chemical composition compared to the underlying material, as aresult of chemical and/or physical processes occurring or performed onthe surface of the pore. For example, treatments that can be usedinclude spontaneous or non-spontaneous oxidation, such as nativeoxidation on silicon, chemical and/or physical post-pore formationtreatments such as deposition of a thin layer of a given material, orsurface activation by plasma, and the like. The exposed surface of oneor more conductive layers, for example, electrode layers, can beselectively passivated or coated with a different metal, eroded,combinations thereof, and the like, by the same chemical-physicaltreatments mentioned above or by electrochemical treatments.Electrochemical treatments can comprise electrodeposition, oxidation,and the like. Passivation can be used if a given electrode is to be usedfor capacitive sensing. A combination of passivated and non-passivatedelectrodes can be used if multiple sensing methods are desired.Electroplating and electro-erosion are ways to grow electrodes insidethe nanopore to decrease the central gap or physical pore size and tocontrol their shape or gap size, not only inwardly, but also outwardly.In some embodiments, an undercut electrode can be made.

FIG. 20 is a top view of nanopore 23 shown in FIG. 19, and depicts thephysical pore diameter 50 and the effective pore diameter 52 resultingfrom the trapping polymer 26 formed on inner sidewall 25 of nanopore 23.In some embodiments, stock particles can be used to at least partiallyfill a nanopore to reduce the translocation rate of a molecule throughthe nanopore. In some embodiments, melted and drawn polymers can beformed and used to reduce the effective pore size of the nanopore.Melted and drawn polymers can be coated on top of a substrate throughwhich a nanopore is formed, and can partially block the nanopore, forexample, at its opening. Polystyrene, polypropylene, polyethylene, andthe like, polymers can be used for such purpose according to variousembodiments. FIG. 20 depicts the physical pore diameter 50 at the levelof electrode 44, and the effective pore diameter 52 that can result froma sensing polymer 48b formed on inner sidewall 25 of nanopore 23.

FIG. 21 is a schematic illustration of a cross-sectional side view of anssDNA molecule 58 being moved through a nanopore 60 according to variousembodiments of the present teachings. Nanopore 60 comprises a firstnucleic acid base binding (affinitive) agent 62 bound to an innersidewall surface of an electrode 63, and a second, different, nucleicacid base binding (affinitive) agent 64 bound to an inner sidewall of adifferent electrode 65. In some embodiments, the exemplary multilayeredstructure 66 can be repeated to provide many layers of electrodessurface modified with different nucleic acid base binding (affinitive)agents. At least one electrode layer can be provided to detect each ofthe four different nucleic acid bases A, C, G, and T. The number ofelectrode layers can be dictated by the sequencing redundancy requiredand the base-resolvability of each individual electrode, for example,the minimum number of electrodes required to resolve the four bases.

FIG. 22 is a top view of nanopore 60 shown in FIG. 21, and shows anelectrode 68 and a counter-electrode 69 spaced apart by dielectricspacers 70 and 71. As can be seen, the electrode 68 can have a muchlarger exposed inner sidewall than counter-electrode 69, for example,from 50% larger to 400% larger, from 100% larger to 300% larger, or from200% larger to 250% larger. In various embodiments, thecounter-electrode can lay on a different layer, or as a counterelectrodeone of the electrodes of a different layer can be used, or even anelectrode not embedded in the nanopore. Also, the function of theelectrode and counterelectrode can also be inverted. As shown in thisdrawing, a counterelectrode can be naked (i.e. without immobilizedpolymers), but it can also have a polymer immobilized on its surface aswell. In summary, a layer can contain one or more electrodes, and one ormore counterelectrodes, with or without immobilized polymers, and theirfunction can be inverted as well as their pairing can be rearrangedduring the operation. Equally, pairing between electrodes andcounterelectrodes on different layers, with or without immobilizedpolymers, is also possible, as well as their functional inversion orpairing rearrangement at run time.

FIG. 23 is a schematic illustration of a cross-sectional side view of anssDNA molecule 78 being moved through a nanopore 80 according to yetother various embodiments of the present teachings. Nanopore 80comprises four different selective nucleic acid base binding(affinitive) agents 82, 84, 86, 88, configured to bind with the fourdifferent nucleic acid bases A, C, G, T. Base binding (affinitive)agents 82, 84, 86, and 88 are bound to four different respectiveelectrodes 92, 94, 96, and 98. FIG. 24 is a top view of the nanopore andmolecule shown in FIG. 23, taken along line 6-6 of FIG. 23, and showingthe arrangement of electrodes 92 and 94. A single counter-electrode 101is provided. Electrodes 92 and 94, and counter-electrode 101, are spacedapart from one another by dielectric spacers 103, 105, and 107. Thedielectric spacers can be made of polymer dielectric materials, forexample, polyimides or fluorinated poly(arylene ethers).

FIG. 25 is a schematic illustration of a cross-sectional side view of anssDNA 111 molecule being moved through a nanopore 112 according tovarious embodiments of the present teachings. Nanopore 112 comprisesselective nucleic acid base binding (affinitive) agents (not shown)bound to surfaces of two-dimensional carbon electrode layers 114, 116,and 118. As shown, electrode layers 114, 116, and 118 are spaced apartfrom one another by dielectric layers comprising silicon nitride or adielectrics polymer. Electrodes 114, 116, and 118 are graphene, which,by virtue of its single layer thickness and its electrical properties,provides superior resolution and electrical transduction. Nanopore 112can also comprise selective nucleic acid base sensing agents (not shown)bound to the exposed atoms of the graphene layers 114, 116, and 118.

According to various embodiments, a nanopore can be provided with ageometry therein that makes the nanopore asymmetrical. An asymmetry canbe provided in the nanopore that causes a molecule, for example, a ssDNAmolecule, to twist as it translocates through the nanopore. The amountof torque applied to the molecule, to move through the pore, can bemeasured. As each base of an ssDNA molecule negotiates past theasymmetry, a distinct torque can be applied and measured, and themolecule can thus be sequenced. In some embodiments, magnetic beads canbe tethered to two opposite ends of a molecule, the two ends can bestretched apart, and the rotation and/or torque resulting from movingeach nucleic acid over or past the asymmetry can be measured, and thebase characteristic of that torque can be determined.

According to various embodiments, a method is provided that comprisesproviding and/or forming a nanopore through a substrate that comprisesat least one layer of graphene. The nanopore can comprise an innersidewall, at least a portion of which comprises an exposed graphenesurface. The exposed graphene surface can be modified by a reaction thatcovalently binds thereto a nucleic acid base binding (affinitive) agent.The binding (affinitive) agent can comprise a carbonyl linkage moietyand a deoxyribonucleotide phosphate. In some embodiments, the phosphatecan comprise a diphosphate or a triphosphate.

FIG. 26 is a schematic illustration of a graphene layer that can be usedas a two-dimensional carbon electrode layer according to variousembodiments of the present teachings. FIG. 27 is an enlarged view of aportion of FIG. 26, showing the average distance between nuclei ofadjacent carbon atoms in the graphene layer. Using graphene can provideatomically thin electrodes. Minimizing electrode thickness can improveresolution and can be used with many of the functionalized nucleic acidbase binding (affinitive) agents described herein. Graphene can be usedfor on-chip integration of both molecular sensing and signal processingelectronics. Further details concerning the use of graphene can be morefully understood with reference to the article of Yu-Ming et al.,Operation of Graphene Transistors at Gigahertz Frequencies, published byIBM T.J. Watson Research Center, Yorktown Heights, N.Y. (Dec. 19, 2008),which is incorporated herein in its entirety by reference.

According to various embodiments, a method of forming a nanoporestructure is provided. FIG. 28 is a cross-sectional side view of ananopore surface modification method that can be used in preparing ananopore according to various embodiments of the present teachings. Themethod can comprise treating a nanopore 120 that is formed through asubstrate 122 comprising at least one layer of silica material 124.Nanopore 120 can comprise an inner sidewall 126 having exposed silanolgroups. The exposed silanol groups can be reacted with anamino-containing alkoxysilane to convert the silanol groups to aminogroups. FIG. 28 depicts such a reaction. As shown silanol groups on theexposed inner sidewall of a silica layer are subject to aminosilylation.

The amino groups thus formed can be reacted with the copolymerizationproduct of an acrylic acid ester of N-hydroxysuccinimde andN,N-dimetjylacrylamide. FIG. 29 is a cross-sectional side view of ananopore surface modification method wherein a trapping or entanglementcopolymer is bonded to the exposed inner surface of nanopore 120 byreacting the reaction product of an acrylic acid ester ofN-hydroxysuccinimde and N,N-dimethyl acrylamide with the amino groups onexposed inner sidewall 126, formed by the method step depicted in FIG.28. The reaction results in the copolymer covalently bonded on exposedinner sidewall 126.

Different molecular weights can be used to fine tune the amount oftrapping or entanglement that can be provided. Molecular weights in therange of from 0.1 to 10 MDa, 0.75 to 5 MDa, or 1 MDa to 2 MDa can beused. For example, a trapping copolymer having a weight of about 1.0 MDacan be used to reduce the effective pore size of a 10 nm nanopore. Insome embodiments, the trapping copolymer can be spin-cast into thenanopore.

In some embodiments, the copolymerized product can be cross-linked byreacting its residual acrylic acid ester of N-hydroxysuccimine with anα,ω-diamino polyol (PEG) to form a cross-linked product that furtherincrease Trapping/entanglement to slow or tension the translocation ofssDNA in the nanopore.

FIG. 30 is a cross-sectional side view of nanopore 120 after asubsequent surface modification wherein the residual acrylic acid esterof N-hydroxysuccinimde in the trapping or entanglement copolymer (asshown in FIG. 29) on exposed inner sidewall 126 is further modified. Asshown in FIG. 30, the further an amino-terminated polyethylene glycolcan be used to react with the unreacted acrylic acid ester ofN-hydroxysuccimine to further increase polymer entanglement. Anα,ω-diamino polyol (PEG), can also be used, in conjunction or alone, toreact with the unreacted acrylic acid ester of N-hydroxysuccimineresulting in a crosslinked 3-D network to improve trappingcharacteristics.

The resulting surface treatment polymer can be useful for slowing downtranslocation of an ssDNA molecule through the nanopore, and forstretching out the ssDNA as it passes through the nanopore. Individualbases of the stretched out ssDNA can thus be more readily detected bydetection moieties in the nanopore, compared to when detection of thebases in a non-stretched conformation.

The esterified acrylic acid can comprise an N-hydroxy succinimide esterof acrylic acid, an N-hydroxy succinimide ester of methacrylic acid, orthe like. The acrylamide can comprise methyl acrylamide, N,N-dimethylacrylamide, or the like.

In some embodiments, a water-soluble capping agent and cross-linker canbe used. In some embodiments, a functional capping agent can be used toprovide not only trapping but also selective nucleic acid basesensitivity. In some embodiments, N-isopropylacrylamide can be used inplace of, or in addition to, N,N-dimethylacrylamide, to provide LCSTcharacteristics.

According to various embodiments, a method is provided for surfacemodification of a nanopore through a substrate that comprises at leastone layer of a noble metal or a noble metal alloy, used as an electrodelayer. The electrode layer can, for example, be electrically connectedto a voltage source and an applied potential can be used that rendersthe electrode an anode. At least a portion of an inner sidewall of thenanopore can be defined by an exposed surface of the at least one layer.In some embodiments, the electrode can comprise gold. According tovarious embodiments, the exposed noble metal or alloy thereof can bereacted, at the exposed surface thereof, with a thiolated compound, suchthat a sulfur linkage to the exposed surface is formed. The thiolatedcompound can comprise a deoxyribonucleotide triphosphate moiety, or thelike. In some embodiments, the method can further comprise reacting thethiolated compound with a deoxyribonucleotide triphosphate prior toreacting the thiolated compound with the exposed surface, and in otherembodiments, such a reaction can be caused after reacting the thiolatedcompound with the exposed surface.

In use, a potential can be applied to the exposed surface to create ananode. In an exemplary embodiment, the noble metal or noble metal alloycomprises gold, for example, pure gold or gold having a purity ofgreater than 95% by weight. For reactions to an exposed gold surface,the thiolated compound can comprise a thiolated polyethylene glycol. Forexample, the thiolated compound can comprise an amino group linked to amercapto group by a poly(ethylene oxide) linker. In some embodiments,the method begins by forming the nanopore before it is treated. Formingcan be by chemical etching, plasma etching, ion etching, laser drilling,micro-machining, or the like.

FIG. 31 is a cross-sectional side view of a nanopore surfacemodification method that can be used in preparing a nanopore accordingto various embodiments of the present teachings. An exposed innersidewall 232 of a gold anode layer 234 of a nanopore 230 is subjected tosurface modification by reaction with a thiolated nucleic acid basebinding agent. One or more different nucleic acid binding (affinitive)agents can be bound to exposed inner sidewall 232 of gold anode 234. Inthe chemical formula shown in FIG. 31, m, n and p can each independentlybe 0, from 1 to 100, from 1 to 50, from 1 to 20, from 1 to 10, or from 1to 5. FIGS. 32A-32F show the chemical structures of six respectivethiolated polyols that can be used in the formation of a nucleic acidbinding agent on inner sidewall surface 232 of gold anode 234 shown inFIG. 31, according to various embodiments.

FIGS. 33A-33D show the chemical structures of four respective nucleicacid binding (affinitive) agents that can be reacted with varioussugar/phosphate moieties and thiolated polyols as described herein toform nucleic acid binding (affinitive) agents being bound by a thiollinkage to inner sidewall surface 232 of gold anode 234 shown in FIG.31. One such sugar phosphate moiety having a base as shown in FIGS.33A-33D is shown in FIG. 34. In some embodiments, each base binding(affinitive) agents can comprise one of the moieties shown in FIGS.33A-33D bound to the remainder of the binding agent through the 9-N atom(as with Ade and Gua) or through the 1-N atom (as with Thy and Cyt). Insome embodiments, functional groups can be bound to the remainder of thebase binding agent with sugar groups, with phosphate groups, or throughpolyA, polyC, polyG, and polyT (U) moieties. In other embodiments, oneof the moieties shown in FIGS. 33A-33D bound to the remainder of thebinding agent through the 9-N atom (as with Ade and Gua) or through the1-N atom (as with Thy and Cyt) can be bound directly to a carbongraphene layer, without a linker.

FIG. 34 shows a deoxyribonucleotide triphosphate that can include one ofthe bases shown in FIGS. 33A-33D in the position indicated, to form anucleic acid binding agent according to various embodiments. The bindingagent can temporarily bind with a complementary base of a target ssDNAstrand as the target strand is moved through nanopore 230 shown in FIG.31. The temporary binding can comprise, for example, the formation ofhydrogen bonds, van der Waals forces, a combination thereof, or thelike, resulting in a change in current that can be detected and used toidentify the base that temporarily bound to the base binding(affinitive) agent.

FIGS. 35 and 36 show a PNA moiety and a DNA moiety, respectively, thatcan be used in forming nucleic acid base binding (affinitive) agentsaccording to other various embodiments of the present teachings.

The nucleic acid base binding (affinitive) agents can be put onrespective electrodes in a controllable manner, using electrochemicalimmobilization. In some embodiments, the binding (affinitive) agents canbe moved by charge attraction/repulsion and covalently bonded intoplace.

According to yet other embodiments of the present teachings, a nanoporeformed through a substrate is provided. The nanopore can comprise aninner sidewall and can have a diameter. The inner sidewall can besurface-modified to have bound to the surface thereof a polymerextending radially inwardly, for example, toward the radial center ofthe nanopore. The polymer can extend inwardly by a distance that is atleast 25% of the length of the diameter, for example, about 35, about45%, or about 55% of the length of the diameter. The inner sidewall canbe surface-modified to have bound to one side of the surface thereof apolymer extending itself across the length of the diameter to theopposite side of the pore. The inner sidewall can also besurface-modified to have multiple points of bonding to the surfacethereof a polymer extending to cover the pore opening at various levels.The diameter can be 100 nm or less, for example, 20 nm or less, or 10 nmor less. The polymer can comprise any of the nanopore surface-modifyingpolymers described herein, for example, the polymer can comprise areaction product of an esterified acrylic acid and an acrylamide, areaction product of a thiolated compound comprising adeoxyribonucleotide phosphate moiety, a reaction product of a carboxylicacid comprising a deoxyribonucleotide phosphate moiety, or the like.

In yet other embodiments of the present teachings, a multilayer nanoporeis provided, that is formed in a substrate. The nanopore can comprise aninner sidewall defined, at least in part, by a first layer. The firstlayer can comprise an exposed surface at the inner sidewall. In someembodiments, the exposed surface can define an electrode, one or morecounter-electrodes, and one or more dielectrics that separate theelectrode from the one or more counter-electrodes. In some embodiments,at least two counter-electrodes are defined at the nanopore innersidewall and each can be surface-modified with a different nucleic acidbase binding (Affinitive) agent covalently bonded thereto at the exposedsurface. With such a configuration, either of at least two differentnucleic acid bases can be identified by the first layer electrodes.Configurations having multiple different layers of electrodes can beused to detect all possible nucleic acid bases and/or to providedetection redundancies useful to verify results.

In use, an electrokinetic force such as an electrophoretic field can beapplied through the nanopore, for example, using an electrode paircomprising an electrode above the nanopore and a counter-electrode belowthe nanopore. The field can be arranged, and of such strength, thatssDNA molecules will translocate through the nanopore from one side tothe other. A reversible field can be configured such that the ssDNA canbe drawn through the nanopore in a first direction, and then through thenanopore in an opposite direction. Such a configuration enablessequencing detection in either and both directions. A back-and-forthapproach can be used to provide redundancies in the base callingsignals, for example, double checking or base calling in forward andreverse directions. Signal processing can be used to throw out badsignals, deconvolute signals, accumulate signals, make base calls,perform combinations of such processes, and the like.

According to various embodiments, non-aqueous solvents can be used as amedia through which target nucleic acids can be moved. Advantageously,when using non-aqueous solvents, no hydrolysis occurs and there is abroader operating voltage window. Non-aqueous solvents can also providelower background noise, a cleaner electrical signal, and a better signalto noise ratio (S/N). The non-aqueous solvent does not necessarily haveto be a good DNA solvent as electrophoretic stretching can make up fornatural relaxation of the target molecule. Moreover, non-aqueoussolvents can be used that have optimal viscosity for DNA translocation.The non-aqueous solvent can be, for example, acetonitrile, DMF, DMSO, orlactam.

Methods of improving oriented movement of a nucleic acid strand throughthe nanopore can be facilitated by adding relatively large molecules, ora macromolecule, to the nucleic acid being sequenced. Suchmacromolecules can, for example, be attached to one end of an ssDNAfragment, resulting in a hydrodynamic drag force in a direction that isopposite the direction of the electric driving force. The macromoleculecan comprise a polymer and can be neutral or charged and its molecularweight does not need to be monodispersed, i.e., Mw/Mn does not have tobe equal to 1. Exemplary macromolecules that can be used for thispurpose include those described, for example, in U.S. Patent ApplicationPublications Nos. U.S. 2008/0241950 A1 to Meagher et al. and U.S.2008/0227211 A1 to Meagher et al., both of which are incorporated hereinin their entireties by reference.

Other references that have devices, systems, methods, and chemistriesthat can be implemented in conjunction with and as part of the presentteachings include U.S. Published Patent Application No. U.S.2008/0187915 A1 to Polonsky et al., publication WO 2008/092760 A1 toPolonsky et al., the article of Morpurgo et al., Controlled fabricationof metal electrodes with atomic separation, American Institute ofPhysics, Volume 74, No. 14, pages 2084-2086 (1999), and the IBM ResearchReport of Polonsky et al., DNA Transistor, IBM Research Division,RC24242, W0704-094 (Apr. 18, 2007), which are incorporated herein intheir entireties by reference.

Other embodiments of the present teachings will be apparent to thoseskilled in the art from consideration of the present specification andpractice of the present teachings disclosed herein. It is intended thatthe present specification and examples be considered exemplary only.

What is claimed:
 1. A method of sequencing a nucleic acid target, themethod comprising: migrating the nucleic acid target through a fluidicnanochannel of an apparatus, the apparatus including: the fluidicnanochannel having a first end, a second end opposite the first end, afirst side, and a second side opposite the first side; a pair ofelectrophoretic electrodes comprising a first electrophoretic electrodeat the first end and a second electrophoretic electrode at the secondend; and a plurality of nano-field effect transistor devices (nanoFETs)disposed in the fluidic nanochannel, wherein the plurality of nanoFETscomprise at least four differently functionalized nanoFETs each having agate electrode functionalized with a different receptor including anucleoside or a protein nucleic acid, the at least four differentlyfunctionalized nanoFETs including a nanoFET functionalized to detectadenine, a nanoFET functionalized to detect cytosine, a nanoFETfunctionalized to detect guanine, and a nanoFET functionalized to detectthymine; wherein a base of the nucleic acid binds with a nanoFET of theplurality of nanoFETs; and detecting the base of the nucleic acid withthe nanoFET bound to the base of the nucleic acid.
 2. The method ofclaim 1, wherein migrating includes applying an electrical field betweenthe pair of electrophoretic electrodes.
 3. The method of claim 2,wherein the electric field is an AC electric field.
 4. The method ofclaim 1, wherein the apparatus further includes a pair of orthogonalelectrodes comprising a first orthogonal electrode at the first side anda second orthogonal electrode at the second side.
 5. The method of claim4, further comprising applying an electrical field using the pair oforthogonal electrodes to influence interaction time between the base andthe nanoFET.
 6. The method of claim 4, wherein the pair of orthogonalelectrodes is part of a set of orthogonal electrodes providing anelectric field having a phase.
 7. The method of claim 6, furthercomprising adjusting the phase of the electric field to move the nucleicacid target.
 8. The method of claim 7, further comprising a second setof orthogonal electrodes providing a separate electric field having asecond phase.
 9. The method of claim 1, wherein the apparatus furtherincludes a memristor network in electrical communication with theplurality of nanoFETs.
 10. The method of claim 9, wherein detecting thebase with the nanoFET includes detecting using the memristor network.11. The method of claim 1, wherein the nucleoside includes adenine,cytosine, guanine, thymine, or uracil.
 12. The method of claim 1,further comprising digesting the nucleic acid target sequentially toprovide a separate nucleotide.
 13. The method of claim 12, furthercomprising driving the separate nucleotide through the channel and intocontact with the plurality of nanoFETs.
 14. The method of claim 13,wherein detecting the base of the nucleic acid target includes detectingthe separate nucleotide.
 15. The method of claim 1, further comprisingbinding the nucleic acid target to a bead prior to migrating.
 16. Themethod of claim 15, further comprising applying an enzyme to the beadprior to migrating.
 17. The method of claim 1, wherein the nanoFETincludes a nanowire transistor.
 18. The method of claim 1, wherein thenanoFET includes a semiconductor-based transistor.
 19. The method ofclaim 1, wherein the fluidic nanochannel is formed from a dielectricmaterial.
 20. The method of claim 1, wherein detecting includesdetecting a current passing through the nanoFET in response to thebinding.